Agradecemos N. Schwedhelm-Domeyer pela excelente assistência técnica, C. Martelo e S. Papiol ajuda com as análises estatísticas. Nosso trabalho é financiado pela Sociedade Max Planck, o Deutsche Forschungsgemeinschaft, o Centro de Microscopia nanoescala e Fisiologia Molecular do Cérebro (CNMPB), Göttingen, Alemanha e pelo GGNB Júnior Grupo Bolsa da Universidade de Göttingen.

CGNs pode ser preparado a partir dia pós-natal (P) 5 filhotes de rato ou P6 filhotes de ratos. Seguimos o protocolo descrito por Bilimoria e colegas, que utiliza um inibidor mitótico para seleccionar para CGNs postmitotic 13. Declaração de Ética: Todos os experimentos envolvendo animais vivos foram realizados de acordo com o protocolo de animais aprovado pela &quot;Verbraucherschutz und Lebensmittelsicherheit&quot; da Baixa Saxônia, Alemanha. Ensaio in vitro: 1. Preparação de DNA plasmídeo, Mídia e amortecedores para o fosfato de cálcio método de transfecção <ol><li> Dissolver DNA plasmídeo em água livre de endotoxina estéril; DMEM (glicose alta); fazer 2,5 M CaCl 2; fazer 2x HBSS (dissolver 4 g de NaCl, 0,1775 g KCl, 0,095 g de Na 2 HPO 4 • 7H 2 O, 0,675 g de glicose e 2,5 g de HEPES em 250 ml de água ultrapura e ajustar o pH a 7,05, 7,08, e 7,11). Observação: Ao se preparara solução 2x HBSS, teste que pH dá melhores resultados em termos de eficiência de transfecção de determinada combinação de plasmídeos. </li></ol> 2. A transfecção de Cultivadas neurônios <img alt="Figura 1" fo:content-width="5in" fo:src="/files/ftp_upload/51070/51070fig1highres.jpg" src="/files/ftp_upload/51070/51070fig1.jpg" /> Figura 1. Fluxograma do axónio vitro e em ensaio de crescimento de dendrites. Cultivadas CGNs (placa de 24 cavidades com lamelas de vidro), isolado a partir de crias de ratos P6, são transfectadas pelo DIV 0 ou 1, com precipitado de ADN contendo um marcador fluorescente transfecção (por exemplo, GFP). Após a fixação e imunocitoquímica, os neurônios são gravadas de forma cega. As imagens são importadas para ImageJ e os processos são medidos. As medições são então processados ​​usando um programa de estatística. <ol><li> CGNs sementes (20 x 10 6 por placa de 24 poços; BME, 10% de soro de vitelo, 2 mM de Penicilina-Streptomycin-Glutamina (PSG), KCl 25 mM) em lavou-ácido nítrico, 12 mm de lamelas de vidro revestidas de poliornitina em uma placa de 24 poços com 500 ul de meio por poço. </li><li> No dia in vitro (DIV) 0 (pelo menos 8 horas após o plaqueamento) ou DIV 1, recolher meios de crescimento e manter a 37 ° C. Lavar neurónios duas vezes com 500 ul de DMEM pré-aquecido e adicionar 500 mL de meio DMEM. </li><li> Colocar os neurónios em incubadora (37 ° C, 5% CO 2) durante 45 min. </li><li> Preparar 40 ul precipitado de ADN para cada poço, misturando: ADN (2-2,5 ug / poço, 10% do DNA total deve ser um marcador de transfecção, por exemplo GFP para visualizar os neurónios transfectadas), água (até 18 mL), adicionar 2 mL de 2,5 M de CaCl2, misturar bem e adicionar 20 ul de HBSS 2X. <ol><li> Incubar ADN precipitado durante 5 minutos à temperatura ambiente. </li></ol></li><li> Adicionar precipitado de ADN a cada poço e incubar durante 18 min neurónios na incubadora. </li><li> Retirar mistura de DMEM / ADN e lavar duas vezes com neurónios 500 ul de DM pré-aquecidoEM. </li><li> Adicionar mídia coletadas a partir do passo 2.2 de volta para os neurônios. Se neurónios será em cultura durante mais do que 3 dias, meios com suplemento de 25 mM de glucose no DIV 3 para reabastecimento da fonte de carbono. </li><li> Depois de 1-5 dias, os neurônios sujeitos a imunocitoquímica utilizando anticorpos GFP. </li><li> Imagem de, pelo menos, 30 neurónios individuais por condição de uma maneira cega usando um microscópio de fluorescência. </li></ol> 3. Meça axônios e dendritos com NeuronJ, uma NIH ImageJ Plugin Importante: Certifique-se de que as imagens são escaladas correctluy usando pixels apropriado: relação mM dependendo ampliação e resolução de imagem. <ol><li> Converter imagens de 8-bit com ImageJ: Abrir imagem, escolha a opção &#39;Imagem&#39; -&gt; &#39;Tipo&#39; -&gt; &#39;8 bits &quot;-&gt;&quot; Salvar imagem. </li><li> Execute NeuronJ plugin e abra a imagem de 8 bits. <img alt="Screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot1.jpg" width="500px" /></li><li> Use a opção &quot;Adicionar traçados &#39;para acompanhar o axônio: click o botão esquerdo do mouse uma vez no início do axônio e mova o mouse ao longo do processo. Dê um clique duplo na ponta da axônio se traço corresponde forma axonal. <img alt="Screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot2.jpg" width="500px" /> Nota: Se o traço sugerido diferem de forma axonal, clique uma vez sobre o processo axonal para ancorar traço, então dê um duplo clique na ponta da axônio. </li><li> Clique em &#39;traçados medida &quot;, escolha a&#39; Mostrar medições rastreamento &#39;opção e pressione&#39; Run &#39;. Medições Axon são todos exibidos em uma nova janela. Para dendrites, escolha a opção &#39;medições grupo de exibição&#39; e pressione &#39;Run&#39;. <img alt="Screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot3.jpg" width="500px" /> Total de medições dendríticas são todos exibidos em uma nova janela. Salvá-los como um arquivo separado que pode ser aberto em qualquer programa de planilha. </li><li> Como alternativa para o traçado manual, use software Fiji: clique com o botão direito no the opção &quot;Reta&quot;, escolha &quot;linha Freehand &#39;, <img alt="Screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot4.jpg" width="500px" /> mantenha o botão esquerdo do mouse pressionado e traçar manualmente o processo, pressione &quot;Ctrl + M &#39;para medir. </li><li> Calcular comprimento / axonal média dendrítica por condição e usar o teste estatístico apropriado. Na eletroporação vivo: </li></ol> 1. Equipamentos e Preparação de Reagentes <ol><li> Você precisa de 30 agulhas G, espaçador (1-2 mm), seringa, redutor de volume morto (DVR), electroporador e tweezertrodes, almofada de aquecimento ou lâmpada de calor infravermelho, luminária e isoflurano. </li><li> Coloque DVR em agulha, em seguida, anexar a agulha na seringa e, finalmente, colocar espaçador na agulha (Figura 2). <img alt="Figura 2" fo:content-width="5in" fo:src="/files/ftp_upload/51070/51070fig2highres.jpg" src="/files/ftp_upload/51070/51070fig2.jpg" /> Figura2. Preparação de agulha. DVR é cortada de uma pipeta de ponta de 200 ul e colocados dentro da agulha, para reduzir o volume morto. Espaçador é derivada a partir de uma ponta de 200 ul de carga e está colocado na extremidade da agulha para regular a profundidade de penetração no cerebelo de aproximadamente 2 mm. Unidades da régua: cm </li><li> Dissolve-se o ADN em PBS/0.03% Fast Green. Observação: Como um marcador de transfecção, é vantajosa a utilização de uma proteína fluorescente que está sob um promotor específico de neurónios (por exemplo, sinapsina) para visualizar apenas neurónios. 25% da quantidade total de plasmídeo deve ser o plasmídeo que codifica para o marcador de transfeco. </li><li> Faça etanol 70%. </li><li> Mistura de volumes iguais de outubro e 30% de sacarose dissolvida em PBS. </li><li> Encher a seringa com 4 ul de DNA (4 mg / mL de DNA de plasmídeo em PBS/0.03% Fast Green). </li></ol> 2. IVE de filhotes de rato Fluxograma de IVE: veja a Figura 3 <p class="jove_content" fo:keep-together.within-Page = &quot;always&quot;&gt; <img alt="Figura 3" fo:content-width="5in" fo:src="/files/ftp_upload/51070/51070fig3highres.jpg" src="/files/ftp_upload/51070/51070fig3.jpg" /> Figura 3. Fluxograma de electroporação in vivo. Filhotes de rato P4 são anestesiados com isoflurano e do plasmídeo de ADN que codifica um marcador de transfecção fluorescente (por exemplo, GFP) é injectado no cerebelo, seguido por exposição a cinco impulsos eléctricos. Cinco dias depois, isolado a GFP-positiva cerebelos são seccionadas e submetidas a imuno-histoquímica. As imagens foram capturadas usando um microscópio confocal e analisados ​​usando o software Imaris. Os dados são processados ​​com um programa de análise estatística. <ol><li> Use filhotes de ratos P4 do albino (Wistar ou Long Evans). </li><li> Anestesiar filhotes (um após o outro) com isoflurano em pequena caixa (por exemplo, caixa de ponteira P1000) com 200 mL de isoflurano (embebido em tecido) para 1-2 min até cachorro não é mais comovente. Tome cuidado para que os filhotes não entrar em contato wom Iiquid isoflurano. Monitore o tempo de perto como filhotes individuais respondem diferentemente à anestesia. </li><li> Esterilizar trás da cabeça de cachorro com etanol 70%. </li><li> Fix cabeça de cachorro entre o polegar eo dedo indicador e usar luminária para localizar cerebelo de filhote albino. O seio transverso demarca nitidamente do mesencéfalo (colículo superior e inferior) dos hemisférios corticais (Figura 3). O cerebelo está localizado ao lado do mesencéfalo e aparece em um tom mais escuro. Use um marcador permanente para indicar o cerebelo com um ponto. Importante: Mantenha filhote em posição fixa! Nota: No caso de anestesia desgastar durante este procedimento, expor filhote ao isoflurano antes de injetar o DNA. </li><li> Inserir a agulha (Figura 3) e, lentamente, injectar 3 ul de DNA para o cerebelo. </li><li> Deixe a solução de DNA para difundir 30-60 seg. <ol><li> Coloque a cabeça do filhote entre tweezertrodes de modo que o pólo negativo entra em contato com a parte de trás da cabeça (cerebeloregião r) e que o pólo positivo em contacto com o lado oposto da cabeça (Figura 3). </li><li> Assunto cachorro para 5 pulsos elétricos. Ajustar a tensão de peso das crias para garantir uma boa eficiência de electroporação, sem comprometer a sua sobrevivência (Tabela 1). <table border="0"><tbody><tr><td> Peso </td><td> Tensão </td><td> Pulso </td><td> Intervalo </td></tr><tr><td> 8-9 g </td><td> 160 V </td><td> 50 mseg </td><td> 950 ms </td></tr><tr><td> 9-10 g </td><td> 165 V </td><td> 50 mseg </td><td> 950 ms </td></tr><tr><td> &gt; 10 g </td><td> 170 V </td><td> 50 mseg </td><td> 950 ms </td></tr></tbody></table> Tabela 1. Eletroporação de P4 filhotes de ratos. </li><li> Vamos recuperar os filhotes na almofada aquecida ou debaixo de uma lâmpada infravermelha. Retornar filhotes para barragem. Importante: Certifique-se de que a fonte de aquecimento não infligir qualquer queimaduras. </li></ol></li><li> Sacrifício filhotes de 5 dias após a electroporação, colocando-os em CO 2 seguido por decapitação. <ol><li> Isolar o cerebelo e a tela para as GFP positivas cerebelos utilizando um microscópio de fluorescência. </li></ol></li><li> Fix cerebelo em PFA a 4% O / N a 4 ° C, em seguida, incuba-se 30% de sacarose a 4 ° C até pia cerebelos de fundo do tubo. </li><li> Incorporar no cerebelo OCT/30% de sacarose e 40 mM cortar secções coronais utilizando um criostato. Nota: A secção cada cerebelo de uma forma cega. </li><li> Assunto seções à imuno-histoquímica utilizando o anticorpo GFP. Contrastante com corante nuclear (DAPI ou Hoechst 33258) e determinar a localização de pelo menos 200 neurônios transfectadas por animal. </li><li> Para uma análise aprofundada, subdividir o IGL em metades, resultando em uma IGL superior de frente para o ML e um IGL menor de frente para a substância branca e contar neurônios GFP positivas que residem em cada semestre. Nota: Contagem neurônios GFP positivas de cada seção de forma cega. </li&gt; </ol> 3. Medindo Dendrite Comprimento, adquirir as imagens da Seção em x, y, z Plano Usando um microscópio confocal Nota: por exemplo, usar 40 imagens para uma seção 40 mM com um z-stwp de 1 mícron. <ol><li> Abrir série de imagens no software, Imaris, para gerar uma imagem 3D dos dendritos. </li><li> Clique no modo &#39;Superar&#39; para ver o neurônio em 3D. <img alt="Screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot5.jpg" width="500px" /></li><li> Selecione &quot;Adicionar novo filamento&quot; e clique em &quot;Ir criação automática &#39;para iniciar o rastreamento semi-automática. <img alt="Screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot6.jpg" width="500px" /> Nota: Analise cada imagem 3D de uma forma cega </li><li> Selecione a guia &#39;Draw&#39; e &#39;autopath&#39;. <img alt="Screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot7.jpg" width="500px" /></li><li> Mova o cursor do mouse no cell corpo e Shift + clique do mouse para a direita para selecionar o corpo celular. Nota: autocalculation por software pode requerer alguns minutos. <img alt="Screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot8.jpg" width="500px" /></li><li> Adicionar caminhos para o filamento (dendritos), usando Shift + clique do mouse para a esquerda. Nota: Os caminhos podem ser visualizados em tempo real. <img alt="Screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot9.jpg" width="500px" /></li><li> Vá para a janela de estatísticas de filamento e clique em, &quot;valores específicos» &#39;detalhadas&#39; e &#39;comprimento Filament dendrito (soma) &quot;para a soma do comprimento total dendrito. <img alt="Screenshot" fo:content-width="5in" src="/files/ftp_upload/51070/screenshot10.jpg" width="500px" /></li><li> Use testes estatísticos apropriados para analisar dados. </li></ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaMKII-NeuroD+Signaling+Pathway+Specifies+Dendritic+Morphogenesis.">CaMKII-NeuroD Signaling Pathway Specifies Dendritic Morphogenesis.</a> Neuron. 41, 229-241 (2004).</li><li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cdc20-APC+ubiquitin+signaling+pathway+regulates+presynaptic+differentiation.">A Cdc20-APC ubiquitin signaling pathway regulates presynaptic differentiation.</a> Science. 326, 575-578 (2009).</li><li>Litterman, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+OBSL1-Cul7Fbxw8+ubiquitin+ligase+signaling+mechanism+regulates+Golgi+morphology+and+dendrite+patterning.">An OBSL1-Cul7Fbxw8 ubiquitin ligase signaling mechanism regulates Golgi morphology and dendrite patterning.</a> PLoS Biol. 9, (2011).</li><li>Kim, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+centrosomal+Cdc20-APC+pathway+controls+dendrite+morphogenesis+in+postmitotic+neurons.">A centrosomal Cdc20-APC pathway controls dendrite morphogenesis in postmitotic neurons.</a> Cell. 136, 322-336 (2009).</li><li>Shalizi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+calcium-regulated+MEF2+sumoylation+switch+controls+postsynaptic+differentiation.">A calcium-regulated MEF2 sumoylation switch controls postsynaptic differentiation.</a> Science. 311, 1012-1017 (2006).</li><li>Vadhvani, M., Schwedhelm-Domeyer, N., Mukherjee, C., Stegmuller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Centrosomal+E3+Ubiquitin+Ligase+FBXO31-SCF+Regulates+Neuronal+Morphogenesis+and+Migration.">The Centrosomal E3 Ubiquitin Ligase FBXO31-SCF Regulates Neuronal Morphogenesis and Migration.</a> PLoS One. 8, (2013).</li><li>Puram, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CaMKIIbeta+signaling+pathway+at+the+centrosome+regulates+dendrite+patterning+in+the+brain.">A CaMKIIbeta signaling pathway at the centrosome regulates dendrite patterning in the brain.</a> Nat. Neurosci. , (2011).</li><li>Jia, Y., Zhou, J., Tai, Y., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPC+channels+promote+cerebellar+granule+neuron+survival.">TRPC channels promote cerebellar granule neuron survival.</a> Nat. Neurosci. 10, 559-567 (2007).</li><li>Huynh, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+isoform-specific+SnoN1-FOXO1+repressor+complex+controls+neuronal+morphogenesis+and+positioning+in+the+mammalian+brain.">An isoform-specific SnoN1-FOXO1 repressor complex controls neuronal morphogenesis and positioning in the mammalian brain.</a> Neuron. 69, 930-944 (2011).</li><li>de la Torre-Ubieta, L., Bonni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+neuronal+polarity+and+morphogenesis+in+the+mammalian+brain.">Transcriptional regulation of neuronal polarity and morphogenesis in the mammalian brain.</a> Neuron. 72, 22-40 (2011).</li><li>Calissano, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+human+insulin-like+growth+factor+I+exerts+a+trophic+action+and+confers+glutamate+sensitivity+on+glutamate-resistant+cerebellar+granule+cells.">Recombinant human insulin-like growth factor I exerts a trophic action and confers glutamate sensitivity on glutamate-resistant cerebellar granule cells.</a> Proc. Natl. Acad. Sci. U.S.A. 90, 8752-8756 (1993).</li></ol>

Os autores declaram não haver interesses financeiros concorrentes.

Vantagens e limitações do descrito, in vitro e in vivo em métodos: CGNs cultivadas de rato e ratos são igualmente adequado para análises morfológicas. Devido ao maior tamanho de cerebelo de rato, o rendimento de CNGs a partir de crias de ratos excede a de filhotes de rato de 3-4x. Além de CGNs, os neurónios corticais e do hipocampo pode ser usado como sistema de cultura, bem. O método de fosfato de cálcio resulta numa baixa (0,01-5%) eficiência de transfecção, ...

O cerebelo é um sistema excelente para estudar mecanismos de crescimento do axônio e migração. O cerebelo tem sido objeto de estudos anatômicos desde o alvorecer da neurociência 1. Microscopia moderna e técnicas de imunohistoquímica têm significativamente ampliado e aperfeiçoado as descobertas iniciais de Santiago, Ramon e Cajal 2-4. Genética do rato e estudos moleculares descobriram fatores de crescimento e de transcrição essenciais no controle do desenvolvimento do cerebelo, o que levou a uma maior compreensão dos acontecimentos cruciais necessários para a ligação adequada dos diferentes tipos de neurônios, incluindo os neurônios granulares do cerebelo (CGNs) 5-7. O cerebelo é um derivado de rhombomere 1 da parte posterior do cérebro em desenvolvimento 8. O lábio rômbico, que é parte do teto do 4 º ventrículo, dá origem a progenitores grânulo de neurônios do cerebelo, que constituem a população neuronal mais numerosos nocerebelo adulto 9. Após a migração rostral, eles se instalarem no anlage cerebelar. Aqui, a mitose dos precursores de grânulos de neurónios conduz à expansão dramática da camada granular externa (EGL), que tem lugar após o nascimento, em roedores. A partir da EGL, os neurônios começam a migrar para o interior através da camada molecular (ML), passando pela camada de células de Purkinje de tomar em última instância, a residir na camada granular interna (IGL 2). Durante esse processo migratório, eles adquirem uma forma bipolar com dois axônios que se estende para o ML. Após mais de migração, o corpo da célula migra longe dos axônios e os dois processos se fundem para formar um bifurcado, axônio em forma de T 10. Posteriormente, esses axônios fasciculada e são referidas como fibras paralelas. Tendo estabelecido no IGL, CGNs crescer dendritos, que formam garras dendríticas para estabelecer sinapses com fibras musgosas. Para examinar os processos fundamentais no cerebelo em desenvolvimento, um combinado in vitro e in vivo approach permite resultados confiáveis ​​e conclusões. CGNs não são apenas os mais numerosos neurônios do cerebelo, mas de todo o cérebro e pode ser cultivado ao elevado grau de pureza 11-13. Na cultura, essa população neuronal altamente homogêneo torna-se rapidamente pós-mitótico e adquire uma morfologia polar com axônios e dendritos facilmente identificáveis. CGNs cultivadas têm provado ser extremamente útil para estudar vários aspectos do desenvolvimento neuronal, incluindo a proliferação de células progenitoras, diferenciação, axonal e desenvolvimento dendrito, a migração neuronal, apoptose e propriedades eletrofisiológicas (14-19 e muitos outros). O uso da manipulação genética ampliou a versatilidade de CGNs cultivadas e permitiu uma visão mais mecanicista nos eventos acima mencionados. A transfecção de neurônios cultivados utilizando fosfato de cálcio de baixa eficiência ou métodos lipofílicos seguido por imunocitoquímica com marcadores de polaridade ou apoiados pelo software de análise de facilitarstados a avaliação de, por exemplo a morfologia de neurónios individuais na cultura neuronal densa. Com esta abordagem, o papel de proteínas de interesse em axónio ou dendrito crescimento pode ser estudada 20-25,26-28. Este sistema de cultivo, porém, é menos útil para analisar a migração neuronal como a migração é muito limitado em culturas de alta densidade e exigiria co-culturas. A análise in vitro de axônio e dendritos crescimento também permite a análise de proteínas interligados de uma via de sinalização que utilizam combinações de interferência de RNA (i), a sobre-expressão ou pequenas moléculas. Para estabelecer a relevância da proteína de interesse em axônio e dendritos regulação do crescimento ou da migração neuronal, a eletroporação in vivo (IVE) técnica permite a análise no córtex cerebelar em desenvolvimento. Devido ao facto de o desenvolvimento do cerebelo em roedores estende caminho para as primeiras duas semanas após o nascimento, o cerebelo representa um accessibestrutura cerebral le para manipulações genéticas para examinar o desenvolvimento de axônios e dendritos, a migração neuronal, sinaptogênese e apoptose 20-24,29,30,26,27,31-34. Além disso, este modelo de sistema também é útil para outros aspectos do desenvolvimento neuronal que exigem o córtex cerebelar intacto como pathfinding axônio, cabeamento e conectividade dos neurônios e interações neurônio-glia em conjunto, este protocolo fornece in vitro e in vivo, para enfrentar uma abordagem complementar sobre morfogênese neuronal e migração.

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			<td height="21" style="height:21px;width:160px;">DMEM</td>
			<td style="width:125px;">Gibco</td>
			<td style="width:88px;">11960-044</td>
			<td style="width:168px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:160px;">BME</td>
			<td style="width:125px;">Gibco</td>
			<td style="width:88px;">41010-026</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:160px;">Insulin</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:88px;">Si-1-4011</td>
			<td style="width:168px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:160px;">Poly-L-Ornithine</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:88px;">P-2533</td>
			<td style="width:168px;"></td>
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		<tr height="25">
			<td height="25" style="height:25px;width:160px;">CaCl2</td>
			<td style="width:125px;">Appli-Chem</td>
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			<td height="42" style="height:42px;width:160px;">Isofluorane</td>
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			<td style="width:168px;"></td>
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			<td height="22" style="height:22px;width:160px;">Tissue-Tek OCT</td>
			<td style="width:125px;">Sakura</td>
			<td></td>
			<td></td>
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		<tr height="44">
			<td height="44" style="height:44px;width:160px;">Material Name</td>
			<td style="width:125px;">Company</td>
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			<td style="width:168px;">Comments (optional)</td>
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			<td height="43" style="height:43px;width:160px;">ECM 830 and tweezertrodes</td>
			<td style="width:125px;">Harvard Apparatus</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
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		<tr height="64">
			<td height="64" style="height:64px;width:160px;">Epifluorescence microscope and camera</td>
			<td style="width:125px;">Nikon</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:160px;">SP2 confocal microscope</td>
			<td style="width:125px;">Leica</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:160px;">ImageJ</td>
			<td style="width:125px;">NIH</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:160px;">Imaris 7.4.2</td>
			<td style="width:125px;">Bitplane, Inc.</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:160px;">GraphPad Prism</td>
			<td style="width:125px;">GraphPad Software, Inc.</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:160px;">MS Excel</td>
			<td style="width:125px;">Microsoft</td>
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			<td style="width:168px;"></td>
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			<td height="22" style="height:22px;width:160px;">Loading tip 1-200 &#181;l</td>
			<td style="width:125px;">Costar</td>
			<td style="width:88px;">4853</td>
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			<td height="22" style="height:22px;width:160px;">Pipette tip 200 &#181;l</td>
			<td style="width:125px;">Sarstedt</td>
			<td style="width:88px;">70.760.502</td>
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			<td height="43" style="height:43px;width:160px;">Microlance 3 needle, 30 gauge</td>
			<td style="width:125px;">BD</td>
			<td style="width:88px;">302200</td>
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			<td height="43" style="height:43px;width:160px;">50 &#181;l gastight Syringe 1705</td>
			<td style="width:125px;">Hamilton</td>
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			<td height="43" style="height:43px;width:160px;">Glass coverslips&#160;</td>
			<td style="width:125px;">Thermo Scientific Menzel Glaeser</td>
			<td>CB00120RA1</td>
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genetic manipulation of cerebellar granule neurons in vitro and in vivo to study neuronal morphology and migration

Eventos do desenvolvimento do cérebro, incluindo a morfogênese neuronal ea migração são altamente processos orquestrado. In vitro e in vivo analisa permitir uma caracterização aprofundada para identificar vias envolvidas nestes eventos. Neurónios de grânulos de cerebelo (CGNs) que são derivadas a partir do cerebelo em desenvolvimento, são um sistema modelo ideal, que permite visualizar as alterações morfológicas. Aqui, descrevemos um método de como manipular geneticamente CGNs e como estudar axono e dendritogenesis de neurônios individuais. Com este método, os efeitos de interferência de ARN, ou a sobre-expressão de moléculas pequenas podem ser comparadas para controlar neurónios. Além disso, o córtex cerebelar é um roedor facilmente acessíveis no sistema in vivo, devido ao seu desenvolvimento pós-natal predominante. Apresentamos também uma técnica de eletroporação in vivo para manipular geneticamente o cerebelo desenvolver e descrever cerebelar posterior análise para avaliar a morfologia neuronal umaª migração.

Para analisar a morfologia de CGNs em resposta a diferentes condições de cultura, que transf os neurónios em DIV 0, como descrito acima. Após a transfecção, que colocado um conjunto de neurónios em meio completo (BME, 10% de soro de vitelo, PSG 2 mM, KCl 25 mM) e um outro conjunto em meio mínimo contendo insulina (BME, glicose 25 mM, PSG a 2 mM, 10 ug / ml de insulina). Nós submetidos os neurônios a imunocitoquímica, utilizando o anticorpo GFP em DIV 1, 2 e 3, seguido pela medição axônios e dendritos para ...

Watch this Scientific Journal Video about Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration at JoVE.com

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Morfogênese neuronal ea migração são eventos cruciais subjacentes ao desenvolvimento do cérebro. Aqui, descrevemos os métodos para manipular geneticamente cultivadas neurônios granulares do cerebelo e do cerebelo em desenvolvimento para a avaliação da morfologia e características migratórias dos neurônios.

Manipulação genética de Cerebelar Granule Neurônios<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em&gt; Estudar Neuronal Morfologia e Migração

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

genetic-manipulation-cerebellar-granule-neurons-vitro-vivo-to-study

Research

JoVE Journal

Neuroscience

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

13.6K visualizações.  Max Planck Institute of Experimental Medicine.  Morfogênese neuronal ea migração são eventos cruciais subjacentes ao desenvolvimento do cérebro. Aqui, descrevemos os métodos para manipular geneticamente cultivadas neurônios granulares do cerebelo e do cerebelo em desenvolvimento para a avaliação da morfologia e características migratórias dos neurônios. 

Vídeo: Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

Análise Sholl automatizado de Morfologia Neuronal Digitized em múltiplas escalas

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

Neurociência

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Lentivírus mediada Manipulação Genética e Visualização de Olfactory neurônios sensoriais In vivo</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

Análise quantitativa de Reposição da vesícula sináptica Piscina em Cultured neurônios granulares do cerebelo usando Dyes FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Rápido Micro-iontoforese de glutamato e GABA: uma ferramenta útil para investigar Integração Synaptic

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted area of the brain, neural stem cells proliferate and constantly generate neuroblasts that migrate tangentially in chains along the rostral migratory stream (RMS) to reach the olfactory bulb (OB). Once in the OB, neuroblasts switch to radial migration and then differentiate into mature neurons able to incorporate into the preexisting neuronal network. Proper neuroblast migration is a fundamental step in neurogenesis, ensuring the correct functional maturation of newborn neurons. Given the ability of SVZ-derived neuroblasts to target injured areas in the brain, investigating the intracellular mechanisms underlying their motility will not only enhance the understanding of neurogenesis but may also promote the development of neuroregenerative strategies.
This manuscript describes a detailed protocol for the transfection of primary rodent RMS postnatal neuroblasts and the analysis of their motility using a 3D in vitro migration assay recapitulating their mode of migration observed in vivo. Both rat and mouse neuroblasts can be quickly and efficiently transfected via nucleofection with either plasmid DNA, small hairpin (sh)RNA or short interfering (si)RNA oligos targeting genes of interest. To analyze migration, nucleofected cells are reaggregated in &#39;hanging drops&#39; and subsequently embedded in a three-dimensional matrix. Nucleofection per se does not significantly impair the migration of neuroblasts. Pharmacological treatment of nucleofected and reaggregated neuroblasts can also be performed to study the role of signaling pathways involved in neuroblast migration.

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Neuroblast migration is a crucial step in postnatal neurogenesis. The protocol described here can be used to investigate the role of candidate regulators of neuroblast migration by employing DNA/small hairpin RNA (shRNA) nucleofection and a 3D migration assay with neuroblasts isolated from the rodent postnatal rostral migratory stream.

Nucleofection de roedores Neuroblastos para estudar neuroblasto Migração<em&gt; In vitro</em

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Induzir Plasticidade de astrocítica Receptores de Manipulação de Neuronal queima de moeda

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

O Sistema Olfativo como um modelo para estudar os padrões de crescimento axonal e Morfologia<em&gt; In Vivo</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

Ex vivo de imagens de pós-natal Cerebelar Granule migração celular Usando confocal Macroscopia

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

Analisando dendríticas Morfologia em Colunas e Camadas

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the co-existence of glial cells and neurons in CGN culture might lead to biases in the accurate assessment of neuronal viability. Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining has been used to measure cell viability by simultaneously evaluating the viable and dead cells. We used FDA-PI double staining to improve the sensitivities of the colorimetric assays and to evaluate neuronal viability in CGNs. Furthermore, we added blue fluorescent DNA stains (e.g., Hoechst) to improve the accuracy. This protocol describes how to improve the accuracy of assessment of neuronal viability by using these methods in CGN culture. Using this protocol, the number of glial cells can be excluded by using fluorescence microscopy. A similar strategy can be applied to distinguish the unwanted glial cells from neurons in various mixed cell cultures, such as primary cortical culture and hippocampal culture.

This protocol describes how to accurately measure neuronal viability using Fluorescein diacetate (FDA) and Propidium Iodide (PI) double staining in cultured cerebellar granule neurons, a primary neuronal culture used as an in vitro model in neuroscience and neuropharmacology research.

Avaliação da Viabilidade Neuronal Utilizando Coloração Dupla de Iodeto de Diacetate-Propidium de Fluoresceína em Cultura de Neurônio de Granulado Cerebelar

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...