Agradecemos Irmgard Weiss-assistência técnica especializada. Este trabalho foi apoiado pelo DFG através do Cluster de excelência para microscopia na escala do nanômetro e fisiologia molecular do cérebro (CNMPB, B1-7, a T.D.).

Foi realizado nenhum estudo com animais vivos. Experimentos envolvendo animais sacrificados para obter culturas foram aprovadas pelas autoridades locais de proteção animal (Tierschutzkommission der Universitätsmedizin Göttingen) sob a aprovação de célula número T10/30. Os experimentos foram realizados com os protocolos aprovados.
1. primário de células Hippocampal cultura
<ol><li>Prepare a cultura de células dissociadas do hipocampo rato em embrionárias dia 1916,17. As células em lamelas de 12 mm, revestidas com polyethyleneimine (PEI) em 24-bem pratos em uma densidade de 50.000-60.000 células/poço da placa. Verifique a densidade utilizando uma célula ótica de contraste de fase e câmara de contagem.</li>
	<li>Cultura os neurônios para 3 dias (dia em vitro (DIV) 3) em uma placa de 24 na incubadora a 37 ° C com 5% de CO2.</li>
	<li>Avalie as lamelas para indicadores de saúde de células usando microscopia de luz transmitida (por exemplo, fase contraste ótica em uma ampliação de 10-20 X). Verifique os seguintes indicadores de saúde: um halo de contraste de fase clara, neuritos sem estruturas frisados e não soma clustering ou agrupamento do axônio.</li>
</ol>2. transfection
Nota: O seguinte protocolo refere-se a um dupla de Transfeccao para 3 poços. No entanto, o protocolo funciona melhor quando quantidades suficientes para 4 poços estão preparadas.
<ol><li>Prepare 500 mL de tampão de transfeccao (274 mM NaCl, 10 mM KCl, 1,4 mM Na2HPO4, glicose de 15mm, 42 mM HEPES) em um Erlenmeyer.<ol>
<li>Dissolva 5,0 g de HEPES em 400 mL de água destilada em um Erlenmeyer, 0,37 g de KCl, 0,095 g de Na2HPO4, 1,35 g de glicose e 8,0 g de NaCl.</li>
		<li>Ajuste o pH a 6,95 com 1 M NaOH, usando um medidor de pH.</li>
		<li>Ajustar o volume com água destilada para 500 mL e verificar o pH usando um medidor de pH.</li>
		<li>Fazer alíquotas de 20-30 mL de tampão de transfecção com os seguintes valores de pH por pipetagem 1 M NaOH para o buffer de transfeccao: 6,96, 6.97, 6,98, 6.99, 7.00, 7.01, 7.02, 7.03, 7.04, 7.05, 7.06, 7,07, 7.08, 7.09, 7.11. Nota: O pH do buffer do transfection é crucial para a eficácia do transfection.</li>
		<li>Para testar o que leva de tampão de Transfeccao para o maior número de células transfectadas, teste cada valor de pH de 6,96 a 7.11. Use o método de transfeccao descrito em 2.2-2.11 e um plasmídeo validado expressando GFP. Determine o número de células transfectadas por lamela para cada valor de pH do tampão de transfeccao avaliar qual tampão funciona melhor.</li>
		<li>Alíquota a reserva com a mais alta eficiência de Transfeccao para tubos de 2 mL microcentrifuga congelar e armazenar os tubos a-20 ° C.</li>
	</ol>
</li>
	<li>Pré-aquecer o soro reduzida meio, meio de cultura celular e água destilada a 37 ° C em banho-maria.</li>
	<li>Prepare a mistura de transfeccao em um tubo de microcentrifugadora de 1,5 mL. Trabalhar sob a capa de fluxo laminar para garantir condições de trabalho estéril.<ol>
<li>Mix de 7,5 µ l de cloreto de cálcio 2 M com 4 µ g de cada DNA livre de endotoxinas (Sinaptofisina-mOrange e mGFP/GFP-Rogdi). Adicione água para atingir um volume total de 60 mL em um tubo de microcentrifugadora de 1,5 mL.</li>
		<li>Adicione 60 mL de tampão de transfeccao à mistura. Para obter os melhores resultados, adicione o buffer de transfeccao gota a gota, agitando a mistura de DNA suavemente sobre o vórtice.</li>
		<li>Incube a temperatura ambiente (RT) por 20 minutos. Evite agitação do tubo de incubação durante o tempo de incubação, colocando o tubo ao lado a capa do fluxo laminar.</li>
	</ol>
</li>
	<li>Sob o capô de fluxo laminar, remover o meio de cultura celular ("meio condicionado") dos poços utilizando uma pipeta de 1000 mL e armazená-lo em um recipiente separado na incubadora.</li>
	<li>Adicione 500 mL de soro reduzida meio a cada poço. Incube as celulas em 37 ° C e 5% de CO2 até que acabe o período de incubação de 20 minutos (etapa 2.3.3).</li>
	<li>Adicione 30 mL de mistura de Transfeccao para cada poço pipetando várias gotas. Descarte o resíduo no fundo do tubo.</li>
	<li>Depois de todos os poços foram fornecidos com a mistura de transfeccao, agite suavemente a placa de 24 para garantir a distribuição da mistura no meio do transfection.</li>
	<li>Incube os poços durante 60 minutos a 37 ° C e 5% de CO2.</li>
	<li>Remova e descarte a mistura de transfeccao e lavá-lo três vezes com meio de cultura celular. Adicione 1 mL de meio de cultura celular a cada poço e incube-os por 30 segundos a RT. remover 750 mL do meio e adicione a mesma quantidade de meio fresco. Repita isso três vezes. Nota: A etapa de lavagem é muito importante. Manter o tempo que cada poço não tem nenhum meio de no mínimo (i. e., remover e substituir por-bem) e adicionar lentamente o meio de lavagem.</li>
	<li>Remova e descarte o meio de cultura celular e adicionar 450 mL do condicionado médio por-bem.</li>
	<li>Deixe os neurônios maduros na incubadora a 37 ° C e 5% CO2 para 10 DIV.</li>
</ol>3. estimulação e captação de Syt1
Nota: O seguinte protocolo aplica-se a absorção de 3 poços. Por despolarização de qualquer outro número de poços, ajuste os montantes em conformidade.
<ol><li>Prepare-se 50 mL de tampão de despolarização 10 x (640 mM NaCl, 700 mM KCl, 10 mM MgCl2, 20mm CaCl2, glicose de 300 mM, 200 mM HEPES, pH 7,4), em um Erlenmeyer. Nota: A reserva de despolarização pode ser mantida a 4 ° C durante várias semanas. Se uma solução não-despolarizantes também é usada, preparar um 10 solução do x Tyrode consistindo de 1290mm NaCl, 50 mM KCl, 10 mM MgCl2, 20mm CaCl2, 300 mM, glicose, pH HEPES 200mm 7.4 fim comparar estimulação induzida por reciclagem com espontânea reciclagem. Após a diluição a 1 x, adicione 1 µM de tetrodotoxina antes do uso para bloquear a geração do potencial de ação.<ol>
<li>Dissolva 1,87 g de NaCl, 2,61 g de KCl, 0,1 g de MgCl2-6 H20, 0,15 g de CaCl2-2 H2O e 3,0 g de glicose-1 H2O e 2,38 g de HEPES em 50 mL de água destilada em um Erlenmeyer. Ajuste o pH com NaOH e estéril-filtrar a solução. Dilua o tampão 01:10 em água destilada a obter uma concentração de 1 x.</li>
	</ol>
</li>
	<li>Prepare o paraformaldeído 4% (PFA) em 1X PBS (pH 7,4) para fixação após a estimulação.<ol>
<li>Para 500 mL de 4% PFA em PBS 1x, dissolver 20 g de paraformaldeído em 450 mL de água destilada H2O. Nota: A solução de aquecimento pode acelerar a dissolução, mas não aqueça a solução acima de 70 ° C, como o PFA pode desintegrar-se. Cuidado: PFA é tóxico, potencialmente carcinogênicos e teratogênicos. Use luvas quando trabalhar com PFA, trabalhar sob a coifa e evitar a ingestão.</li>
		<li>Deixe a solução esfriar a RT e adicionar 50 mL de solução estoque de PBS x 10. Ajuste o pH para 7,4 com NaOH/HCl usando um medidor de pH.</li>
	</ol>
</li>
	<li>Pré-aqueça 600 mL de tampão de despolarização 1x e 10 mL de meio de cultura celular para 37 ° C em banho-maria.</li>
	<li>Adicione 1 mL de anticorpo anti-Syt1 de rato (clone 604.2) para o 1x buffer de despolarização e vortex durante 10 segundos.</li>
	<li>Remova e descarte o meio de cultura celular de células. Adicione 200 mL da mistura despolarização-anticorpo para cada bem e incubar por 5 minutos a 37 ° C e 5% CO2 na incubadora.</li>
	<li>Remova e descarte a mistura de despolarização-anticorpo e lavá-lo três vezes com meio de cultura celular. Adicione 1 mL de meio de cultura celular a cada poço e incube por 30 segundos a RT. remover 750 mL do meio e adicione a mesma quantidade de meio fresco. Repeti três vezes.</li>
	<li>Remova e descarte o meio de cultura celular e adicionar 300ml de 4% PFA em 1X PBS. Incubar durante 20 minutos a 4 ° C.</li>
	<li>Lavar três vezes por 5 minutos, cada um com PBS 1x. Nota: O protocolo pode ser pausado aqui.</li>
</ol>4. imunocitoquímica
<ol><li>Prepare 50 mL de tampão de bloqueio. Nota: Bloqueio de buffer pode ser mantido a-20 ° C por vários meses.<ol>
<li>Dissolva 2,5 g de sacarose e 1 g de albumina de soro bovino (BSA) em 5 mL de solução estoque de PBS x 10. Adicione 1,5 mL da solução detergente 10%. Agite a solução até que todos os componentes corretamente se dissolveram e adicionar água destilada H2O até atingir um volume final de 50 mL. Alíquota da solução e congelar as alíquotas para armazenamento.</li>
	</ol>
</li>
	<li>Dilua o anticorpo secundário, fluoróforo acoplados (dirigido contra as espécies de Syt1-anticorpo primárias) em 200 mL de tampão de bloqueio em cada poço a uma diluição de 1: 1000.</li>
	<li>Remova e descarte a PBS 1x de cada bem que contém uma lamela.</li>
	<li>Adicionar 200 mL de mistura de tampão-anticorpo para cada poço de bloqueio e incubar durante 60 minutos a RT Cuidado: Porque os anticorpos secundários são sensíveis à luz, todos os passos em frente devem ser realizados no escuro.</li>
	<li>Após a incubação, lave as células três vezes por 5 minutos com 1 mL de 1X PBS.</li>
	<li>Incorpore as lamelas em corrediças do microscópio com o medium de incorporação.<ol>
<li>Adicione uma gota de 7 mL de incorporação médio da lâmina de microscópio. Remova a lamela a placa de 24 por levantá-lo com uma seringa e agarrando-o com a pinça. Cuidado: Células na superfície da lamela são facilmente danificadas, então fórceps deve ser manuseado com cuidado.</li>
		<li>Mergulhe a lamela água destilada para remover a PBS e seque-o cuidadosamente, tocando uma borda de um tecido mole.</li>
		<li>Inverter a lamela sobre a incorporação da gota média, para que a superfície carregando as células enfrenta a lâmina de microscópio, incorporando, assim, as células no meio de encastre.</li>
	</ol>
</li>
	<li>Deixe os slides para secar sob o capô para 1-2 h (cobri-los para evitar a exposição à luz) e armazená-los em uma caixa de slide de microscópio a 4 ° C. Nota: O protocolo pode ser pausado aqui.</li>
</ol>5. microscópico análise
<ol><li>Depois de secam as lamelas, colocá-los sob o microscópio com o objectivo e a câmera.</li>
	<li>Ajuste o tempo de exposição para cada canal para que alguns pixels são superexpostas para garantir o máximo da distribuição dos valores de cinza. Nota: Enquanto o tempo de exposição pode variar entre os canais, deve ser constante para um canal para assegurar a comparabilidade entre as lamelas.</li>
	<li>Adquira imagens de multi-canal para 10 regiões de interesse (ROIs) por lamela. Verifique se o ROI contém processos axonal a partir de um neurônio transfectado, verificando a GFP-fluorescência, que deve ser punctate.</li>
</ol>6. análise estatística
<ol><li>Exporte as imagens como arquivos. tif. Carregar as imagens para OpenView18 clicando em arquivo | Arquivo de imagem de carga.</li>
	<li>Escolha a imagem Sinaptofisina-mOrange como canal 1, a imagem Rogdi-GFP/mGFP como o canal 2 e a imagem de fluorescência Alexa647 como canal 3.</li>
	<li>Limiar de ROIs.<ol>
<li>Clique em análise | Área de lugar sobre a partitura. Escolha os valores de limiar e intensidade de Delta para que após a inspeção visual, fluorescência difusa é excluída, deixando apenas punctate sinais na imagem do canal 1 (representando Sinaptofisina-mOrange fluorescência). Manter o mesmo limiar para todas as imagens.</li>
	</ol>
</li>
	<li>Transferi estes ROIs ao correspondente canal 2 (fluorescência de GFP-Rogdi/mGFP) e 3 (Syt1-fluorescência) de cada área de lamela, clicando em executar agora. Nota: O ROIs só deve ser considerado se a célula é duplo-transfectadas. Neste método, o mOrange e GFP-fluorescência deve ser claramente visível.</li>
	<li>Salve os dados no editor de registo de análise clicando em dados de Log.</li>
	<li>Abra o editor de registro de análise sob a guia do Windows e copiar os valores para cada canal. Cole os valores para os canais separados em uma planilha.</li>
	<li>Determine a intensidade média de fluorescência do Syt1-canal no ROIs em ambas as condições de transfeccao (GFP-Rogdi e mGFP).</li>
	<li>Aplica testes estatísticos adequados, tais como o teste t de Student para determinar diferenças significativas.</li>
</ol>

<ol>
	<li>Koh, T., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptotagmin+I,+a+Ca2++sensor+for+neurotransmitter+release.">Synaptotagmin I, a Ca2+ sensor for neurotransmitter release.</a> Trends in Neurosciences. 26, 413-422 (2003).</li><li>Chapman, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Does+Synaptotagmin+Trigger+Neurotransmitter+Release.">How Does Synaptotagmin Trigger Neurotransmitter Release.</a> Annual Review of Biochemistry. 77, 615-641 (2008).</li><li>Perin, M. S., 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=Structural+and+Functional+Conservation+of+Synaptotagmin+(p65)+in+Drosophila+and+Humans.">Structural and Functional Conservation of Synaptotagmin (p65) in Drosophila and Humans.</a> Journal of Biological Chemistry. 266, 615-622 (1991).</li><li>Matteoli, M., Takei, K., Perrin, M. S., S&#252;dhof, T. C., De Camilli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exo-endocytotic+Recycling+of+Synaptic+Vesicles+in+Developing+Processes+of+Cultured+Hippocampal+Neurons.">Exo-endocytotic Recycling of Synaptic Vesicles in Developing Processes of Cultured Hippocampal Neurons.</a> Journal of Cell Biology. 117, 849-861 (1992).</li><li>Ko, J., 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=Neuroligin-1+performs+neurexin-dependent+and+neurexin-independent+functions+in+synapse+validation.">Neuroligin-1 performs neurexin-dependent and neurexin-independent functions in synapse validation.</a> The EMBO Journal. 28, 3244-3255 (2009).</li><li>Shinoda, 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=BDNF+enhances+spontaneous+and+activity-dependent+neurotransmitter+release+at+excitatory+terminals+but+not+at+inhibitory+terminals+in+hippocampal+neurons.">BDNF enhances spontaneous and activity-dependent neurotransmitter release at excitatory terminals but not at inhibitory terminals in hippocampal neurons.</a> Frontiers in Synaptic Neuroscience. 6, 27 (2014).</li><li>Ivanova, D., 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=Synaptic+activity+controls+localization+and+function+of+CtBP+1+via+binding+to+Bassoon+and+Piccolo.">Synaptic activity controls localization and function of CtBP 1 via binding to Bassoon and Piccolo.</a> The EMBO Journal. 34, 1056-1077 (2015).</li><li>Crawford, D. C., Ramirez, D. M. O., Trauterman, B., Monteggia, L. M., Kavalali, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+molecular+impairment+of+spontaneous+neurotransmission+modulates+synaptic+efficacy.">Selective molecular impairment of spontaneous neurotransmission modulates synaptic efficacy.</a> Nature Communications. 8, 1-14 (2017).</li><li>Riemann, D., Wallrafen, R., Dresbach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Kohlsch&#252;tter-T&#246;nz+syndrome+associated+gene+Rogdi+encodes+a+novel+presynaptic+protein.">The Kohlsch&#252;tter-T&#246;nz syndrome associated gene Rogdi encodes a novel presynaptic protein.</a> Scientific Reports. 7, (2017).</li><li>Kim, M., 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=Rogdi+Defines+GABAergic+Control+of+a+Wake-promoting+Dopaminergic+Pathway+to+Sustain+Sleep+in+Drosophila.">Rogdi Defines GABAergic Control of a Wake-promoting Dopaminergic Pathway to Sustain Sleep in Drosophila.</a> Scientific Reports. 7, 1-14 (2017).</li><li>Schossig, A., Wolf, N. I., Kapferer, I., Kohlsch&#252;tter, A., Zschocke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epileptic+encephalopathy+and+amelogenesis+imperfecta:+Kohlsch&#252;tter-T&#246;nz+syndrome.">Epileptic encephalopathy and amelogenesis imperfecta: Kohlsch&#252;tter-T&#246;nz syndrome.</a> Euopean Journal of Medical Genetics. 55, 319-322 (2012).</li><li>Kraszewski, K., 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=Synaptic+Vesicle+Dynamics+in+Living+Cultured+Hippocampal+Neurons+Visualized+with+CY3-Conjugated+Antibodies+Directed+against+the+Lumenal+Domain+of+Synaptotagmin.">Synaptic Vesicle Dynamics in Living Cultured Hippocampal Neurons Visualized with CY3-Conjugated Antibodies Directed against the Lumenal Domain of Synaptotagmin.</a> Journal of Neuroscience. 15, 4328-4342 (1995).</li><li>Willig, K. I., Rizzoli, S. O., Westphal, V., Jahn, R., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STED+microscopy+reveals+that+synaptotagmin+remains+clustered+after+synaptic+vesicle+exocytosis.">STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis.</a> Nature. 440, 935-939 (2006).</li><li>Petkova, A., Goedecke, N., Korte, M., Dresbach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroligins+mediate+presynaptic+maturation+through+brain-derived+neurotrophic+factor+signaling.">Neuroligins mediate presynaptic maturation through brain-derived neurotrophic factor signaling.</a> bioRxiv. , 262246 (2018).</li><li>Fuchs, C., 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=GABA(A)+receptors+can+initiate+the+formation+of+functional+inhibitory+GABAergic+synapses.">GABA(A) receptors can initiate the formation of functional inhibitory GABAergic synapses.</a> European Journal of Neuroscience. 38, 3146-3158 (2013).</li><li>Dresbach, T., 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=Functional+regions+of+the+presynaptic+cytomatrix+protein+Bassoon:+Significance+for+synaptic+targeting+and+cytomatrix+anchoring.">Functional regions of the presynaptic cytomatrix protein Bassoon: Significance for synaptic targeting and cytomatrix anchoring.</a> Molecular and Cellular Neuroscience. 23, 279-291 (2003).</li><li>Seibenhener, M. L., Wooten, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Culture+of+Hippocampal+Neurons+from+Prenatal+Mice.">Isolation and Culture of Hippocampal Neurons from Prenatal Mice.</a> Journal of Visualized Experiments. (65), 4-9 (2012).</li><li>Tsuriel, S., 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=Local+sharing+as+a+predominant+determinant+of+synaptic+matrix+molecular+dynamics.">Local sharing as a predominant determinant of synaptic matrix molecular dynamics.</a> PLOS Biology. 4, 1572-1587 (2006).</li><li>Iwabuchi, S., Kakazu, Y., Koh, J., Goodman, K. M., Harata, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+Synaptic+Vesicle+Recycling+Using+FM+Dyes+During+Evoked,+Spontaneous,+and+Miniature+Synaptic+Activities.">Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities.</a> Journal of Visualized Experiments. (85), 1-10 (2014).</li><li>Villarreal, S., Lee, S. H., Wu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Synaptic+Vesicle+Endocytosis+in+Cultured+Hippocampal+Neurons.">Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons.</a> Journal of Visualized Experiments. (127), 1-8 (2017).</li><li>Kavalali, E. T., Jorgensen, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+presynaptic+function.">Visualizing presynaptic function.</a> Nature Neuroscience. 17, 10-16 (2014).</li><li>Opazo, F., 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=Limited+Intermixing+of+Synaptic+Vesicle+Components+upon+Vesicle+Recycling.">Limited Intermixing of Synaptic Vesicle Components upon Vesicle Recycling.</a> Traffic. 11, 800-812 (2010).</li><li>Wollebo, H. S., Woldemichaele, B., White, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+transduction+of+neuronal+cells.">Lentiviral transduction of neuronal cells.</a> Methods in Molecular Biology. 1078, 141-146 (2013).</li><li>Yang, X., Kaeser-Woo, Y. J., Pang, Z. P., Xu, W., S&#252;dhof, 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=Complexin+Clamps+Asynchronous+Release+by+Blocking+a+Secondary+Ca2++Sensor+via+Its+Accessory+&#945;+Helix.">Complexin Clamps Asynchronous Release by Blocking a Secondary Ca2+ Sensor via Its Accessory &#945; Helix.</a> Neuron. 68, 907-920 (2010).</li><li>Wittenmayer, 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=Postsynaptic+Neuroligin1+regulates+presynaptic+maturation.">Postsynaptic Neuroligin1 regulates presynaptic maturation.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 13564-13569 (2009).</li><li>Lazarevic, V., Schone, C., Heine, M., Gundelfinger, E. D., Fejtova, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+Remodeling+of+the+Presynaptic+Cytomatrix+upon+Homeostatic+Adaptation+to+Network+Activity+Silencing.">Extensive Remodeling of the Presynaptic Cytomatrix upon Homeostatic Adaptation to Network Activity Silencing.</a> Journal of Neuroscience. 31, 10189-10200 (2011).</li><li>Kraszewski, K., Daniell, L., Mundigl, O., De Camilli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobility+of+synaptic+vesicles+in+nerve+endings+monitored+by+recovery+from+photobleaching+of+synaptic+vesicle-associated+fluorescence.">Mobility of synaptic vesicles in nerve endings monitored by recovery from photobleaching of synaptic vesicle-associated fluorescence.</a> Journal of Neuroscience. 16, 5905-5913 (1996).</li><li>Hua, Y., Sinha, R., Martineau, M., Kahms, M., Klingauf, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+origin+of+synaptic+vesicles+undergoing+evoked+and+spontaneous+fusion.">A common origin of synaptic vesicles undergoing evoked and spontaneous fusion.</a> Nature Neuroscience. 13, 1451-1453 (2010).</li><li>Han, W., 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=N-Glycosylation+Is+Essential+for+Vesicular+Targeting+of+Synaptotagmin+1.">N-Glycosylation Is Essential for Vesicular Targeting of Synaptotagmin 1.</a> Neuron. 41, 85-99 (2004).</li><li>Kwon, S. E., Chapman, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosylation+is+dispensable+for+sorting+of+synaptotagmin+1+but+is+critical+for+targeting+of+SV2+and+synaptophysin+to+recycling+synaptic+vesicles.">Glycosylation is dispensable for sorting of synaptotagmin 1 but is critical for targeting of SV2 and synaptophysin to recycling synaptic vesicles.</a> Journal of Biological Chemistry. 287, 35658-35668 (2012).</li><li>Afuwape, O. A. T., Wasser, C. R., Schikorski, T., Kavalali, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+vesicle+pool-specific+modification+of+neurotransmitter+release+by+intravesicular+free+radical+generation.">Synaptic vesicle pool-specific modification of neurotransmitter release by intravesicular free radical generation.</a> The Journal of Physiology. 595, 1223-1238 (2017).</li><li>Sara, Y., Virmani, T., De&#225;k, F., Liu, X., Kavalali, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+isolated+pool+of+vesicles+recycles+at+rest+and+drives+spontaneous+neurotransmission.">An isolated pool of vesicles recycles at rest and drives spontaneous neurotransmission.</a> Neuron. 45, 563-573 (2005).</li><li>Wilhelm, B. G., Groemer, T. W., Rizzoli, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+same+synaptic+vesicles+drive+active+and+spontaneous+release.">The same synaptic vesicles drive active and spontaneous release.</a> Nature Neuroscience. 13, 1454-1456 (2010).</li><li>Bacci, 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=Chronic+blockade+of+glutamate+receptors+enhances+presynaptic+release+and+downregulates+the+interaction+between+synaptophysin-synaptobrevin-vesicle-associated+membrane+protein+2.">Chronic blockade of glutamate receptors enhances presynaptic release and downregulates the interaction between synaptophysin-synaptobrevin-vesicle-associated membrane protein 2.</a> Journal of Neuroscience. 21, 6588-6596 (2001).</li><li>Piccoli, G., 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=LRRK2+Controls+Synaptic+Vesicle+Storage+and+Mobilization+within+the+Recycling+Pool.">LRRK2 Controls Synaptic Vesicle Storage and Mobilization within the Recycling Pool.</a> Journal of Neuroscience. 31, 2225-2237 (2011).</li><li>Tracy, T. E., Yan, J. J., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+knockdown+of+AMPA+receptors+reveals+a+trans-synaptic+signal+for+presynaptic+maturation.">Acute knockdown of AMPA receptors reveals a trans-synaptic signal for presynaptic maturation.</a> The EMBO Journal. 30, 1577-1592 (2011).</li><li>Truckenbrdot, S., Viplav, A., Jaehne, S., Vogts, A., Denker, A., Wildhagen, H., Fornasiero, E. F., Rizzoli, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ageing+synaptic+vesicles+are+inactivated+by+contamination+with+SNAP25.">Ageing synaptic vesicles are inactivated by contamination with SNAP25.</a> bioRxiv. , 172239 (2017).</li></ol>

Os autores não têm nada para divulgar.

Existem três ensaios rotineiramente usados para estudar a vesícula sináptica (SV) reciclagem. Os dois primeiros incluem o uso de um) fluorescente styryl corantes como FM1-43 (que incorporar em membranas, são retomadas em organelas durante a endocitose e são liberados após a exocitose); e b) marcados fluorescentemente proteínas recombinantes do SV (que, em cima de superexpressão, incorporam as máquinas de reciclagem proteicas). Se o anexadas fluorophores alterar sua fluorescência dependendo do pH, pode ser usado...

Estudando a vesícula sináptica reciclagem é importante em determinar como alteram Propriedades pré-sináptica, durante a plasticidade sináptica, ou em resposta a perturbação da função sináptica. Estudando Synaptotagmin-1 (Syt1) captação de anticorpo fornece um método para medir a quantidade de reciclagem de SV. Syt1 é uma proteína de SV-associado que atua como um sensor de Ca2 + e é necessária para os liberação do neurotransmissor1,2. É uma proteína transmembranar com um domínio citoplasmático do C-terminal fora o SV e um domínio N-terminal do luminal dentro do SV3. Durante a exocitose, o domínio luminal de Syt1 torna-se exposto ao meio externo. Para este meio externo, acrescentamos anticorpos dirigidos contra o domínio citoplasmático, que se torna interiorizado durante a endocitose. Estes anticorpos podem ser que também pre-conjugados com fluorophores ou immunostained com anticorpos secundários4,5,6,7. A intensidade da fluorescência do immunosignal resultante é proporcional à quantidade de reciclagem de SV. Esta abordagem pode ser usada para determinar o SV constitutiva e induzida por despolarização reciclagem6,8.
Ensaios de absorção de Syt1 podem ser realizados após a transferência de genes mediada por vírus para praticamente todas as células no prato ou transfection esparso de um pequeno número de células. Nosso método combina o ensaio com esparso transfection duplo de neurônios hippocampal preliminares usando o fosfato de cálcio9. Nós usamos uma proteína recombinante marcador conhecida a acumular-se no presynapses, Sinaptofisina fluorescente etiquetada, localizar terminais pré-sináptica e overexpress nossa proteína de interesse, Rogdi. Isto permite-nos testar ou não Rogdi alvos funcionais sinapses e afeta SV reciclagem. O gene que codifica a Rogdi foi originalmente identificado em uma tela para mutantes de Drosophila, caracterizados pela memória prejudicada10. Em humanos, mutações no gene Rogdi causam uma doença rara e devastadora, chamada síndrome de Kohlschütter-Tönz. Os pacientes sofrem de malformações do esmalte dentário e pharmacoresistant epilepsia psicomotoras atrasos; no entanto, a Localização subcellular do produto gene permaneceu evasivo11. Assim, o ensaio de absorção de Syt1 forneceu evidência chave para localização de GFP-etiquetado Rogdi às sinapses funcionais9.
Esta técnica de captação tem diversos benefícios. Primeiro, SV reciclagem pode ser observado tanto em tempo real através da realização de7,de imagens ao vivo12e após fixação6,9 , medindo a intensidade da fluorescência do rótulo Syt1 fluorescência. Além disso, foram desenvolvidas diversas variantes de Syt1 anticorpo. Existem variantes sem marcas de formatação que podem ser rotuladas com um anticorpo secundário, seguindo um protocolo padrão de imunocoloração após a fixação e variantes pre-conjugadas com um rótulo de fluorescência já anexado. Finalmente, baseados em anticorpo fluorescência é vantajosa devido a grande seleção de corantes secundários ou conjugados em comercialmente disponíveis que pode ser usado.
Quando fixação e imunocoloração os neurônios, é também possível mancha para proteínas adicionais e executar análise colocalization. Isto pode ajudar a determinar onde eles estão localizados em relação à reciclagem SVs. A intensidade do rótulo da fluorescência é a medida direta da quantidade de SV reciclagem. Além disso, os anticorpos seletivamente rotulam Syt1 contendo estruturas, resultando em alta especificidade e de fluorescência do pouco fundo4. Protocolos de estimulação diferentes também podem ser usados, tais como buffers de despolarização ou estimulação elétrica protocolos9,12,13,14. No entanto, reciclagem de SV basal pode ser medida sem estimular as culturas neuronal15.
Nosso método trata especificamente a absorção de anticorpo Syt1 nos neurônios duplo-transfectadas com anticorpo secundário immunolabeling após a fixação. No entanto, nos referimos a todas as variantes rotineiramente utilizadas do ensaio em nossa discussão para dar aos espectadores a oportunidade de adaptar o protocolo para necessidades específicas.

<table>
	<tbody>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7030-50g</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C3306-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>CoolSNAP HQ2</td>
			<td>Photometrics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dH2O</td>
			<td>Invitrogen</td>
			<td>15230</td>
			<td></td>
		</tr>
		<tr>
			<td>DABCO</td>
			<td>Merck</td>
			<td>8.03456.0100</td>
			<td></td>
		</tr>
		<tr>
			<td>donkey anti mouse Alexa 647</td>
			<td>Jackson-Immunoresearch</td>
			<td>715605151</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>41966</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes</td>
			<td>Eppendorf</td>
			<td>30120094</td>
			<td></td>
		</tr>
		<tr>
			<td>multiwell 24 well</td>
			<td>Fisher Scientific</td>
			<td>087721H</td>
			<td></td>
		</tr>
		<tr>
			<td>tube (50 mL)</td>
			<td>Greiner Bio-One</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS superior</td>
			<td>BiochromAG</td>
			<td>S0615</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Merck</td>
			<td>1,083,421,000</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Invitrogen</td>
			<td>14170</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Hera Cell 150 (Inkubator)</td>
			<td colspan="2">ThermoElectron Corporation</td>
			<td></td>
		</tr>
		<tr>
			<td>KCL</td>
			<td>Sigma-Aldrich</td>
			<td>P9541-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamin</td>
			<td>Gibco</td>
			<td>25030</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Honeywell</td>
			<td>M0250-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides</td>
			<td>Fisher Scientific</td>
			<td>10144633CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mowiol4-88</td>
			<td>Calbiochem</td>
			<td>475904</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>BioFroxx</td>
			<td>1394KG001</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>BioFroxx</td>
			<td>5155KG001</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Invitrogen</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>OpenView Experiment Analysis Application</td>
			<td>Free software, see comments</td>
			<td></td>
			<td>written by Noam E. Ziv, Technion &#8211; Israel Institute of Technology, Haifa, Israel</td>
		</tr>
		<tr>
			<td>PBS (10x)</td>
			<td>Roche</td>
			<td>11666789001</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimem</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td></td>
		</tr>
		<tr>
			<td>Penstrep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Sigma</td>
			<td>P6148-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>safety hood</td>
			<td>ThermoElectron</td>
			<td></td>
			<td>Serial No. 40649111</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>neoFroxx</td>
			<td>1104kg001</td>
			<td></td>
		</tr>
		<tr>
			<td>Synaptotagmin1</td>
			<td>Synaptic Systems</td>
			<td>105311</td>
			<td>mouse monoclonal; clone 604.2</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Merck</td>
			<td>1,086,031,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Genius 3&#160;</td>
			<td>IKA</td>
			<td>3340001</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>GFL</td>
			<td>1004</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Observer. Z1&#160;</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an optical assay for synaptic vesicle recycling in cultured neurons overexpressing presynaptic proteins

Nos terminais de nervo pré-sináptica ativo, vesículas sinápticas passam por ciclos de exo e endocitose. Durante a reciclagem, os domínios luminal de proteínas transmembrana SV tornam-se expostos na superfície da célula. Uma destas proteínas é Synaptotagmin-1 (Syt1). Um anticorpo dirigido contra o domínio luminal de Syt1, uma vez adicionado ao meio de cultura, é retomado durante o ciclo exo-endocytotic. Esta absorção é proporcional à quantidade de SV reciclagem e pode ser quantificada através de imunofluorescência. Aqui, nós combinamos Syt1 absorção de anticorpos com duplo transfeccao de neurônios hippocampal culturas. Isso permite que nós (1) localizar sites pré-sináptica baseados na expressão do marcador pré-sináptica recombinante Sinaptofisina, (2) determinar sua funcionalidade usando Syt1 absorção e (3) caracterizar a segmentação e os efeitos de uma proteína de interesse, GFP-Rogdi.

Um resultado esperado desta abordagem é localizar aproximadamente 50 duplo-transfectadas neurônios por lamela em uma densidade de 50.000 neurônios por bem. O axónio de cada neurônio é esperado para mostrar vários hotspots de acumulação Sinaptofisina fluorescente etiquetado, indicando aglomerados ofSVs. Em sites pré-sináptica funcionais, o sinal de Sinaptofisina recombinante colocalizes com punctate Syt1 fluorescência. Usando a transfeccao duplo, ou GFP-Rogdi como a proteína d...

Watch this Scientific Journal Video about An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins at JoVE.com

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

Descrevemos um ensaio óptico para vesícula sináptica (SV) reciclagem em neurônios cultivados. Combinar este protocolo com duplo transfeccao de expressar uma proteína de interesse e o marcador pré-sináptica nos permite localizar sites pré-sináptica, sua vesícula sináptica, capacidade de reciclagem e determinar o papel da proteína de interesse.

Um ensaio óptico para vesícula sináptica reciclagem nos neurônios cultivados superexpressão de proteínas pré-sináptica

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...

an-optical-assay-for-synaptic-vesicle-recycling-cultured-neurons

Research

JoVE Journal

Neuroscience

7.4K visualizações.  University Medical Centre Göttingen. Descrevemos um ensaio óptico para vesícula sináptica (SV) reciclagem em neurônios cultivados. Combinar este protocolo com duplo transfeccao de expressar uma proteína de interesse e o marcador pré-sináptica nos permite localizar sites pré-sináptica, sua vesícula sináptica, capacidade de reciclagem e determinar o papel da proteína de interesse.

Vídeo: An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

Estudando Pools vesículas Synaptic usando fotoconversão de Corantes Styryl

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

Neurociência

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

Recuperação de fluorescência Após Fotodegradação (FRAP) de fluorescência Proteínas Tagged em espinhas dendríticas dos neurônios do hipocampo Cultivadas

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

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

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

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

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

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

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

Análise de Morfologia Spine dendríticas em neurônios cultivados CNS

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

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 ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

Induzir o crescimento dendrítico no cultivadas neurônios simpáticos

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

Difusão lateral e Exocitose de proteínas de membrana em neurônios cultivados avaliada através da recuperação de fluorescência e fluorescência perda de fotobranqueamento

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using styryl FM dyes, which reveal membrane turnover. Conventional protocols for the use of FM dyes were designed for analyzing neurons following stimulated (evoked) synaptic activity. Recently, protocols have become available for analyzing the FM signals that accompany weaker synaptic activities, such as spontaneous or miniature synaptic events. Analysis of these small changes in FM signals requires that the imaging system is sufficiently sensitive to detect small changes in intensity, yet that artifactual changes of large amplitude are suppressed. Here we describe a protocol that can be applied to evoked, spontaneous, and miniature synaptic activities, and use cultured hippocampal neurons as an example. This protocol also incorporates a means of assessing the rate of photobleaching of FM dyes, as this is a significant source of artifacts when imaging small changes in intensity.

We describe the use of styryl FM dyes to image synaptic vesicle recycling in functional nerve terminals. This protocol can be applied not only to evoked, but also spontaneous and miniature synaptic activities. The protocol expands the variety of synaptic events that can be effectively evaluated.

Exame do Synaptic Vesicle Reciclagem Usando FM Corantes Durante Evocado, espontânea, e as atividades de Synaptic miniatura

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

Quantificação de Endosome e Moosidades de Lisosoma em Neurônios Cultivados Usando Sondas Fluorescentes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.