Gostaríamos de agradecer aos Profs. Huibert Mansvelder e Bert Sakmann para amplo suporte, Dr. Marcel Oberlaender para discussões frutíferas e proporcionando rastreamento neuronal, e Brendan Lodder para assistência técnica. Os dados foram adquiridos utilizando o ntrode VI para LabVIEW, generosamente fornecidos pela R. de Bruno (Columbia Univ., NY, EUA). Esta pesquisa foi apoiada pela Max Planck Society e do Centro Bernstein de Neurociência Computacional, Tuebingen (financiado pelo Ministério Federal Alemão de Educação e Pesquisa (BMBF; FKZ: 01GQ1002)) (RTN), Centro de Pesquisa e Neurogenomics Cognitiva (CNCR) , Neuroscience Campus Amsterdam (NCA), o financiamento para CPJdK (NWO-ALW # 822.02.013 e ENC-rede # P3-c3) ea VU University Amsterdam.

1. Preparação do animal Todos os procedimentos experimentais são realizados de acordo com a lei holandesa e após avaliação por um comitê de ética local no VU University Amsterdam, Holanda. <ol><li> Anestesiar uma ratazana Wistar (P25-P45, ♂ / ♀) com isoflurano (2-3% em oxigénio) e, subsequentemente, com uretano (20% em 0,9% de NaCl, 1,6-1,7 g / kg) por injecção intraperitoneal. Avaliar a profundidade da anestesia, monitorando retirada pitada, reflexos das pálpebras, e os movimentos de vibrissas. </li><li> Administrar uma dose adicional de uretano (10% da dose inicial) no caso de o animal não chega a profundidade da anestesia suficiente ou quando espasmos vibrissas são observadas durante o curso da experiência. </li><li> Colocar o animal anestesiado na armação estereotáxica equipado com uma almofada de aquecimento, inserir sonda de temperatura rectal e manter a temperatura do corpo do animal, a 37,5 ± 0,5 ° C por uma almofada de aquecimento. </li><li&gt; Posicione a cabeça do animal no quadro estereotáxico; cortar o cabelo no topo da cabeça com uma tesoura para tornar a pele visível. </li><li> Injectar 100 ul de 1% de lidocaína (em NaCl a 0,9%) por via subcutânea para a anestesia local no sítio da operação e esperar 3-5 min. Remover a pele que cobre a superfície do crânio por corte na direcção caudal para rostral e limpar o tecido restante. </li><li> Limpe o crânio extensivamente com NaCl 0,9% e compressas absorventes. </li><li> Determinar as coordenadas da área do alvo no hemisfério esquerdo (para o córtex somatossensorial primário (S1): 2.5 mm posterior e 5,5 mm lateral ao bregma). Marque a localização no crânio com uma caneta marcador pele cirúrgico. </li><li> Raspe suavemente o osso por uma broca de dentista da região de interesse até o osso torna-se transparente e os vasos sanguíneos são claramente visíveis. </li><li> Fazer uma 0,5 mm x 0,5 milímetros craniotomia por corte de uma janela no crânio diluído com um bisturi (# 11), evitando danos à dura-mátere os vasos sanguíneos. Opcional: marcar as bordas da craniotomia usando uma caneta marcador cirúrgico da pele para melhorar a visibilidade. </li><li> Aplicar uma fina camada de supercola sobre o crânio seco e, em seguida, cimento dental para construir uma casa de banho (ou seja, câmara de registo) que envolve a craniotomia. </li><li> Aparar os bigodes no lado contralateral a exatamente 5 mm da folículo triz. Opcional: destacar os bigodes aparados com rímel preto. </li></ol> 2. Juxtasomal Recordings e biocitina Labeling <ol><li> Faça pipetas pedaço de vidro de borosilicato com diâmetro de ponta dentro de ~ 1 mícron obter eletrodos com 3-5 resistência mohms. Nota: A morfologia pipeta ideal para a gravação juxtasomal é um afunilamento gradual delgado, um baixo ângulo de cone, e um ~ 1 um ponta (Figura 1). </li><li> Dependendo da profundidade de gravação (com respeito à superfície pial), as dimensões do atarraxamento do eléctrodo de registo deve ser ajustado. Criticamente importante é que tele cone diâmetro deve ser inferior a 75 um do ponto de entrada no cérebro para evitar danos mecânicos ou tensão para a área de gravação. Isto indica que para as gravações corticais superficiais, um cone de 300-500 um, com um diâmetro exterior de máximo 75 um é requerido (Figura 1A) enquanto que as gravações de camadas corticais profundas relativamente requerem um maior afunilamento de 1.500-1.800 mM, novamente com exterior máxima diâmetro de 75 mM (a 1.800 mM a partir da ponta do eletrodo). </li><li> Carregar a pipeta patch com campainha normal de rato (NRR) suplementado com 2% de biocitina (ver Tabela 1 para as soluções e reagentes) e montar a pipeta patch em um estágio cabeça fixa a um micromanipulador. </li><li> Definir o ângulo de suporte de eléctrodo a 34 ° em relação ao plano sagital para alvejar especificamente a coluna D2 de S1 rato. </li><li> Conecte o palco cabeça a um amplificador em modo bridge (ou seja, grampo atual). </li><li> Posicione o patchpipeta em estreita proximidade com a craniotomia. Encha a banheira com 0,9% de NaCl e determinar a resistência do eletrodo que deve ser entre 3-5 mohms, avaliada pela aplicação de um pulso quadrado com injeção de corrente positiva (1 nA, 200 ms on / off). </li><li> Estabelecer sobrepressão de 100-150 mbar e avançar a pipeta patch no 1 mícron passos durante a aplicação corrente positiva como pulsos quadrados (1 NA, 200 ms on / off). Monitorar a mudança de resistência robusto ao estabelecer contato com a dura-máter. Neste ponto, definir as coordenadas do micromanipulador de &quot;zero&quot; para permitir que as medições de profundidade precisas. </li><li> Avanço em 1 mícron passo-mode até a pipeta de patch penetra na dura-máter indicado por uma queda súbita na resistência do eletrodo. Retire a pressão realização da pipeta. </li><li> Procure por unidades individuais, enquanto avançam em 1 mM passos. Monitorar a resistência do eletrodo de forma contínua através da aplicação de 200 ms on / off pulsos. Um aumento no Neossolo resistência do eletrodoaliado indica a proximidade de um único neurónio. Avance até o eletrodo potencial de ação positiva (AP) formas de onda de ~ 2 mV são registradas (Figuras 2A e 2B). </li><li> Opcional: Grave o padrão spiking espontânea do neurônio e determinar spiking, por exemplo, caudal desviando bigodes individuais para 200 milissegundos em um ângulo de 3,3 ° usando um dispositivo piezoelétrico 18 evocava-triz. </li><li> Avançar o eletrodo até que a resistência é de 25-35 mohms e spikes têm amplitudes de 3-8 mV para a obtenção de condições ótimas de enchimento juxtasomal. Inicie o preenchimento juxtasomal aplicando pulsos quadrados de corrente positiva (1 nA, 200 ms on / off). Lentamente e gradualmente aumentar a corrente em passos de 0,1 nA enquanto se monitoriza a forma de onda de AP e de frequência (Figuras 2A e 2B). </li><li> Monitorar a abertura da membrana como um claro aumento na freqüência de AP durante a fase do pulso em bloco (Figura 2C </strong&gt;). A forma de onda de pico durante o enchimento mostra uma largura aumentada e reduzida após-hiperpolarização (Figuras 2C e 2D). Os parâmetros adicionais incluem aumento de ruído ou um pequeno (1-5 mV) mudança negativa DC. </li><li> Aumentar ou diminuir a corrente (1 nA) ao preencher a manter infusão biocitina estável. Reduzir ou parar os pulsos de corrente em cima súbito aumento da freqüência de AP para evitar toxicidade por excesso de influxo de íons extracelulares, tais como íons de sódio. Nota: cada neurônio respondem de forma diferente à injeção de corrente e juxtasomal parâmetros de rotulagem precisa ser ajustada dependendo das condições de gravação individuais. </li><li> Acompanhar de perto o sinal após a interrupção da injeção de corrente. A forma de onda de pico depois de uma sessão de enchimento é geralmente ampliado e mostra uma forte redução após hiperpolarização. Espere para a recuperação do neurônio, que é aparente quando a forma de onda de pico retorna às suas propriedades originais (ou seja, presença denormal após-hiperpolarização, Figura 2). </li><li> Repita biocitina enchendo sessões após a recuperação completa do neurônio para aumentar a carga biocitina para a melhoria da qualidade de coloração. </li><li> Recolha a pipeta patch passos de 1 mM até que a amplitude do pico diminui para reduzir qualquer tensão mecânica para a célula. Espere pelo menos 1 hora para o biocitina para difundir no meio intracelular, enquanto monitorando de perto a temperatura do corpo do animal e respirar. </li></ol> 3. Irrigando o animal e retirar o cérebro <ol><li> Preparar a configuração da perfusão, enxágüe e pré-carregar os tubos com 0,9% de NaCl. </li><li> Coloque o animal em uma bandeja cirúrgica e prenda com fita rotulagem padrão. Assegurar profundidade suficiente da anestesia; pitada pé e reflexos palpebrais deve estar ausente. </li><li> Faça uma incisão medial para lateral (5-6 cm) através da parede abdominal logo abaixo da caixa torácica e proceder em sentido póstero-anterior para expor o esterno.Puxe o esterno anteriormente e separe cuidadosamente o fígado a partir do diafragma. </li><li> Fazer uma pequena incisão no diafragma, cortar as nervuras inferiores e continuar a incisão ao longo de todo o comprimento da cavidade abdominal para expor o coração. </li><li> Retire o pericárdio. </li><li> Insira a agulha para o ventrículo esquerdo e fazer uma incisão no átrio direito. Perfundir com 0,9% de NaCl (~ 8 ml / min) até descoloração do fígado é completa. </li><li> Mudar a infusão de 4% paraformaldeído (PFA), até que a rigidez da frente pata e maxilar inferior é aparente. </li><li> Decapitar rato usando um par de tesouras </li><li> Retire os músculos do pescoço restantes e expor o crânio completamente. </li><li> Posicione a tesoura no tronco cerebral no lado dorsal e cortar o osso cuidadosamente ao longo da sutura sagital, mantendo a posição dorsal. </li><li> Retirar os ossos de ambos os lados da sutura sagital para expor o cérebro, utilizando um fórceps. Remova cuidadosamente a dura para evitar dAmage. </li><li> Insira com cuidado uma espátula sem corte para o lado ventral do cérebro e remover o cérebro suavemente. </li><li> Pós-corrigir toda a noite cérebro em PFA a 4 ° C. Desligue o cérebro para tampão fosfato 0,05 M (PB) e armazenar a 4 ° C. </li></ol> 4. Fatiar o cérebro em seções tangenciais <ol><li> Pegue o cérebro fora do 0,05 M PB e colocá-lo em um papel de filtro enfrentando anterior. Use uma lâmina afiada para cortar o cerebelo ao longo do plano coronal e separar os hemisférios através do corte ao longo do plano sagital médio. </li><li> Aplicar supercola na plataforma de montagem e montar o hemisfério esquerdo do seu plano sagital com anterior virado para a direita. Fixe a plataforma de montagem em um ângulo de 45 ° em um vibratome e submergir o cérebro em 0,05 M PB. </li><li> Prenda uma lâmina de barbear no vibratome e certifique-se que o primeiro contato com a superfície do cérebro está no meio do avião ântero-posterior do hemisfério. Corte de 24 100 pmseções e coletá-los em uma placa de 24 poços contendo 0,05 M PB. </li></ol> 5. Procedimentos histológicos <ol><li> Realizar protocolos histológicos para a coloração do citocromo-oxidase e 24 a-biotina-peroxidase de avidina método de acordo com os métodos anteriormente descritos 25. Opcional: visualizar biocitina usando conjugados fluorescentes avidina / estreptavidina-Alexa. Este adicionalmente permite-coloração dupla com técnicas de rastreamento retrógrado ou anterógrado. </li><li> Lavar as secções de 5 x 5 minutos com 0,05 M PB e preparar o tetracloridrato de 3, 3&#39;--diaminobenzidina (DAB) de solução para a coloração de citocromo-oxidase para visualizar barris na camada 4 (L4) de S1 contendo (ver Tabela 2 para soluções e reagentes). Incubar secções 6-12 do pia na solução pré-aquecida para 30-45 min a 37 ° C. </li><li> Enxágüe seções com 0,05 M PB por 6 x 5 min e saciar a atividade da peroxidase endógena pela incubação todas as seções em 3% H 2 </sub&gt; O 2 em 0,05 M PB, durante 20 minutos à temperatura ambiente (RT). </li><li> Enxágüe seções com 0,05 M PB por 5 x 10 min. Incubar em secções ABC-solução (ver Tabela 3 para as soluções e os reagentes) durante a noite a 4 ° C. </li><li> Enxágüe seções com 0,05 M PB por 5 x 10 min e preparar a solução DAB para visualizar o neurônio cheio de biocitina (ver Tabela 4 para soluções e reagentes). Incubar secções em solução filtrada por 45-60 minutos à temperatura ambiente. </li><li> Enxágüe seções com 0,05 M PB por 5 x 10 min. Seções montar em lâminas de microscópio e lamínula com Mowiol (ver Tabela 5 para soluções e reagentes). </li><li> Determine qualidade rotulagem usando microscopia de luz. </li></ol>

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P., Huber, D., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+activity+in+barrel+cortex+underlying+vibrissa-based+object+localization+in+mice.">Neural activity in barrel cortex underlying vibrissa-based object localization in mice.</a> Neuron. 67 (6), 1048-1061 (2010).</li><li>Veinante, P., Deschenes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+study+of+motor+cortex+projections+to+the+barrel+field+in+rats.">Single-cell study of motor cortex projections to the barrel field in rats.</a> J. Comp. Neurol. 464 (1), 98-103 (2003).</li><li>Boudewijns, Z. 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=Layer-specific+high-frequency+action+potential+spiking+in+the+prefrontal+cortex+of+awake+rats.">Layer-specific high-frequency action potential spiking in the prefrontal cortex of awake rats.</a> Front. Cell. Neurosci. 7, 99 (2013).</li><li>Oberlaender, M., Bruno, R. M., Sakmann, B., Broser, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmitted+light+brightfield+mosaic+microscopy+for+three-dimensional+tracing+of+single+neuron+morphology.">Transmitted light brightfield mosaic microscopy for three-dimensional tracing of single neuron morphology.</a> J. Biomed. Opt. 12 (6), 064029 (2007).</li><li>Boudewijns, Z. 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=Semi-automated+three-dimensional+reconstructions+of+individual+neurons+reveal+cell+type-specific+circuits+in+cortex.">Semi-automated three-dimensional reconstructions of individual neurons reveal cell type-specific circuits in cortex.</a> Commun. Integr. Biol. 4 (4), 486-488 (2011).</li><li>Bruno, R. M., Hahn, T. T., Wallace, D. J., de Kock, C. P., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+experience+alters+specific+branches+of+individual+corticocortical+axons+during+development.">Sensory experience alters specific branches of individual corticocortical axons during development.</a> J. Neurosci. 29 (10), 3172-3181 (2009).</li><li>Schubert, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observing+without+disturbing:+how+different+cortical+neuron+classes+represent+tactile+stimuli.">Observing without disturbing: how different cortical neuron classes represent tactile stimuli.</a> J. Physiol. 581 (1), 5 (2007).</li><li>Neumann, E., Kakorin, S., Toensing, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+electroporative+delivery+of+drugs+and+genes).">Fundamentals of electroporative delivery of drugs and genes).</a> Bioelectrochem. Bioenerg. 48 (1), 3-16 (1999).</li><li>Haas, K., Sin, W. C., Javaherian, A., Li, Z., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+electroporation+for+gene+transfer+in+vivo.">Single-cell electroporation for gene transfer in vivo.</a> Neuron. 29 (3), 583-591 (2001).</li></ol>

Os authros tem nada a revelar.

O método permite a gravação de juxtasomal em ação vivo potencial spiking de unidades individuais de todo condições comportamentais (anestesiados, acordado ou se movendo livremente fixo-cabeça) com a opção do neurônio gravado para hoc classificação pós tipo de célula e / ou reconstrução 3D-rotulando biocitina. A principal vantagem é a obtenção de parâmetros fisiológicos na configuração extracelular (assim não invasiva), sendo ainda capaz de marcar o neurónio i...

As redes neurais consistem em vários tipos de células, caracterizado por as propriedades morfológicas e fisiológicas altamente específicos 1-7. Como consequência, os tipos de células individuais de realizar tarefas especializadas no seio da rede (ver, por exemplo Gentet et al. 8 e Burgalossi et al. 9). Estamos apenas começando a entender as funções específicas do tipo de célula através de redes neuronais e muito ainda está para ser descoberto. Para este fim, muitos laboratórios são adoptar abordagens experimentais que permitem a análise de propriedades morfológicas da mesma população neuronal do que os parâmetros fisiológicos foram obtidos 1,10-15. Aqui, demonstramos o juxtasomal técnica de marcação, que envolve 16,17 registos electrofisiolicos utilizando pipetas de patch convencionais na configuração extracelular (assim não invasiva) em combinação com a electroporação do neurónio gravado com biocitina. Ogrande vantagem desta abordagem é que a natureza não invasiva assegura que a acção potencial cravação de neurónios individuais é gravado sem alterar (por exemplo, diálise) o conteúdo intracelular da célula. Seguido por eletroporação, a abordagem juxtasomal oferece a opção de identificação post hoc celular e reconstrução de ligação de função (fisiologia) para estrutura (morfologia). Normalmente, a reconstrução morfológica envolve a reconstrução da morfologia dendrítica e axonal, que pode ser estendido para quantificação da coluna e / ou Bouton densidades ou mesmo reconstrução da morfologia neuronal com resolução nanométrica usando microscopia eletrônica. A técnica de gravação juxtasomal pode ser usado para in vivo gravações de vários tipos de células em todo camadas corticais ou em áreas sub-corticais em uma variedade de espécies, embora a maioria dos estudos têm aplicado a técnica em pequenos roedores, como camundongos ou ratos. Nossa pesquisa está focada na gravação e rotulagem de neurôniosde rato córtex somatossensorial primário (S1) e envolve a identificação visual de neurônios registrados 18, reconstruções dendríticas, em combinação com o registro preciso de um quadro de referência padronizada para fazer engenharia reversa de redes corticais 4,19 e reconstrução detalhada da arquitetura axonal caracterizar tipo específico de células locais e projeção de longo alcance como alvo 20. Comparado com in vivo as técnicas de gravação alternativos (intracelulares ou de células inteiras), as gravações juxtasomal são relativamente estáveis ​​e, portanto, pode ser aplicado em estados comportamentais incluindo anestesiados 21,22, sedado 14, 23 animais fixo-cabeça, ou mesmo livremente em movimento acordado 9 . Aqui, vamos mostrar rotulagem juxtasomal em S1 de um rato anestesiado-uretano, embora ressaltamos a aplicabilidade geral desta técnica para muitas preparações de escolha.

<table border="0" cellpadding="0" cellspacing="0" width="784">
	<tbody>
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			<td>National Instruments, Austin, TX, USA</td>
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			<td>R. Bruno, Columbia Univ., NY, USA</td>
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			<td height="21" style="height:21px;width:224px;">(Labview acq. software)</td>
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			<td height="21" style="height:21px;width:224px;">Cytochrome C from equine heart</td>
			<td style="width:275px;">Sigma</td>
			<td style="width:119px;">C2506</td>
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			<td style="width:275px;">Sigma</td>
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			<td height="21" style="height:21px;width:224px;">DAB</td>
			<td style="width:275px;">Sigma</td>
			<td style="width:119px;">D5637</td>
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			<td height="21" style="height:21px;width:224px;">H2O2</td>
			<td style="width:275px;">Boom</td>
			<td style="width:119px;">7047</td>
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			<td style="width:275px;">Vector</td>
			<td style="width:119px;">PK6100</td>
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			<td height="21" style="height:21px;width:224px;">Triton X100</td>
			<td style="width:275px;">Sigma</td>
			<td style="width:119px;">T9284</td>
			<td></td>
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			<td height="21" style="height:21px;width:224px;">Urethane</td>
			<td style="width:275px;">Sigma</td>
			<td style="width:119px;">U2500</td>
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			<td height="21" style="height:21px;width:224px;">Isoflurane</td>
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			<td style="width:119px;">45.112.110</td>
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			<td height="21" style="height:21px;width:224px;">Lidocaine</td>
			<td style="width:275px;">Sigma</td>
			<td style="width:119px;">L5647</td>
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			<td height="21" style="height:21px;width:224px;">Simplex rapid dental cement</td>
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			<td>ACR308/ACR924</td>
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			<td height="21" style="height:21px;width:224px;">PFA</td>
			<td style="width:275px;">Merck Millipore</td>
			<td style="width:119px;">8187151000&#160;</td>
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			<td height="21" style="height:21px;width:224px;">Trizma base</td>
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			<td style="width:119px;">T4661</td>
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			<td height="21" style="height:21px;width:224px;">Mowiol 4-88</td>
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			<td height="21" style="height:21px;width:224px;">Analytical grade glycerol</td>
			<td style="width:275px;">Fluka</td>
			<td style="width:119px;">49767</td>
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			<td height="21" style="height:21px;width:224px;">HEPES</td>
			<td style="width:275px;">Sigma</td>
			<td>H3375</td>
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			<td height="21" style="height:21px;width:224px;">NaCl</td>
			<td style="width:275px;">Sigma Aldrich</td>
			<td style="width:119px;">31434</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:224px;">KCl</td>
			<td style="width:275px;">Sigma Aldrich</td>
			<td style="width:119px;">60130</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:224px;">CaCl</td>
			<td style="width:275px;">Sigma Aldrich</td>
			<td style="width:119px;">22,350-6</td>
			<td></td>
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			<td height="21" style="height:21px;width:224px;">MgCl2</td>
			<td style="width:275px;">Fluka</td>
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</table>

juxtasomal biocytin labeling to study the structure-function relationship of individual cortical neurons

O córtex cerebral é caracterizada por múltiplas camadas e muitos tipos de células distintas que em conjunto, como uma rede de responsáveis ​​por várias funções cognitivas superiores, incluindo a tomada de decisões, o comportamento sensório-guiada ou memória. Para entender como essas redes neuronais complexas executar tais tarefas, um passo crucial é determinar a função (ou atividade elétrica) de tipos de células individuais dentro da rede, preferencialmente quando o animal está realizando uma tarefa cognitiva relevante. Além disso, é igualmente importante para determinar a estrutura anatômica da rede e da arquitetura morfológica dos neurônios individuais para permitir a engenharia reversa da rede cortical. Avanços técnicos disponíveis hoje permitem gravar a atividade celular em acordada, comportando animais com a valiosa opção de post hoc identificar os neurônios registrados. Aqui, nós demonstrar a técnica de rotulagem juxtasomal biocitina, que envolve a ação de gravação potenciômetroal spiking na configuração extracelular (ou solta-patch) usando pipetas de patch convencionais. A configuração de gravação juxtasomal é relativamente estável e aplicável em condições comportamentais, incluindo anestesiado, sedado, acordado fixo-cabeça, e até mesmo no animal se movendo livremente. Assim, este método permite que liga tipo de célula ação específica potencial spiking durante comportamento animal para a reconstrução dos neurônios individuais e, em última análise, todo o microcircuito cortical. Neste vídeo manuscrito, vamos mostrar como os neurônios individuais na configuração juxtasomal pode ser rotulado com biocitina em ratos anestesiados com uretano para identificação post hoc e reconstrução morfológica.

O conhecimento detalhado sobre a estrutura 3D de neurônios individuais é crucial para elucidar os princípios organizacionais das redes neuronais. Nosso método consiste em um gasoduto para alcançar rotulagem biocitina alta qualidade a partir de uma preparação in vivo, permitindo assim a classificação post hoc neuronal e reconstrução detalhada do dendrítica e arquitetura axonal de neurônios individuais em alta resolução. Dependendo da qualidade da rotulagem juxtasomal, os neurónios são r...

Watch this Scientific Journal Video about Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons at JoVE.com

Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

Para entender a estrutura das redes neuronais, caracterização funcional e morfológica de neurônios individuais é uma necessidade. Aqui, nós demonstramos rotulagem juxtasomal biocitina, que permite que registros eletrofisiológicos na configuração extracelular, mas mantendo a capacidade de rotular intracelular do neurônio para a reconstrução post hoc de dendrítica e arquitetura axonal.

Juxtasomal biocitina Labeling para estudar a função Estrutura de Relacionamento Individual neurônios corticais

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive ...

juxtasomal-biocytin-labeling-to-study-structure-function-relationship

Research

JoVE Journal

Neuroscience

13.5K visualizações.  VU University Amsterdam.  Para entender a estrutura das redes neuronais, caracterização funcional e morfológica de neurônios individuais é uma necessidade. Aqui, nós demonstramos rotulagem juxtasomal biocitina, que permite que registros eletrofisiológicos na configuração extracelular, mas mantendo a capacidade de rotular intracelular do neurônio para a reconstrução post hoc de dendrítica e arquitetura axonal. 

Vídeo: Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

Laser Microdissecção Captura de Drosophila Neurônios periféricos

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Neurociência

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

Bilaminar Co-cultura de neurônios de rato Primária Cortical e Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

DII-rotulagem de neurônios DRG para estudar em uma ramificação axonal Preparação Monte Whole of Mouse Embryonic Spinal Cord

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

Vivo em dois fótons de imagem de experiência dependentes de alterações moleculares em neurônios corticais

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

Um Método para High Fidelity Optogenetic Controle de Individual Piramidal neurônios<em&gt; In vivo</em

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

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

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

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

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

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 ...

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

Rotulagem diferencial de superfície celular e internalizado Proteínas após Anticorpo Alimentação da Vivo Cultivadas neurônios

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Captura Laser Microdissection - uma demonstração do Isolamento de Pessoa dopamina Neurônios e região ventral tegmental Entire

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present&#160;a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

A imuno-histoquímica e Rotulagem múltipla com anticorpos da espécie hospedeira Mesmas para Estudar Adulto Hippocampal Neurogênese

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Quantificação de filamentosas actina (F-actina) puncta no Rat neurônios corticais

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...