Os autores agradecem RIKEN para financiamento. Agradecemos também Cagri Yalgin, Caroline Delandre, e Jay Parrish para discussões sobre protocolos de genética e imuno-histoquímica.

1.Preparação de reagentes <ol><li> Prepare Ca + + livre HL3.1 salina 28. <ol><li> Em mM: 70 NaCl, KCl 5, 20 MgCl 2, 10 NaHCO 3, 5 HEPES, 115 sacarose, trealose e 5; pH 7.2. Filtro de esterilizar e armazenar a 4 ° C. Nota: Ca + + livre solução impede a contração muscular durante a dissecção. </li></ol></li><li> Faça poli-L-lisina (PLL) lamínulas. <ol><li> Dissolver 100mg PLL em água 4.2ml e fazer alíquotas em tubos de Eppendorf 300 ul e congelar a -20 ° C. </li><li> Antes de lamínulas de revestimento, primeiro degelo uma alíquota, trazê-lo até 10ml em DDH 2 O e adicionar 20μl do Kodak Photo-Flo, a concentração final PLL é de 0,7 mg / ml. </li><li> Lamínulas submergir por 30 minutos em solução PLL, remover e seca, em seguida, lavar rapidamente com água e seca. Repita duas vezes. Lamínulas tratados durar aproximadamente um mês. </li></ol></li></ol> 2. Cruzamentos genéticos <ol><li> To gerar clones MARCM. Aqui está um exemplo cruz usando um driver de pan-DA 11,27: FRT2A x hsFLP; Gal4 109 (2) 80, UAS-mCD8:: GFP; banheira Gal80 FRT2A/SM5-TM6B </li><li> Para gerar FLP-out clones. Aqui está uma cruz exemplo usando uma classe de driver específico IV-8: ppk-Gal4 x yw, hsFLP; UAS-FRT-CD2, + y-stop-FRT-mCD8:: GFP </li></ol> 3. Colheita de embriões <ol><li> Mantenha cruza Drosophila em um frasco coletor a 25 ° C e coletar embriões em um suco de maçã espalhar placa agar 29 com uma fina camada de pasta de levedura 30,31. </li></ol> 4. Tratamento de choque térmico de embriões <ol><li> Coloque um prato vazio em oposição à placa de ágar suco de maçã sobre a qual os embriões tenham sido estabelecidas. Selo em torno destas duas placas com Parafilm (Fig. 2). </li><li> Durante o choque de calor ª submergir,placa e no banho de água e segure-o para baixo, usando um peso de metal. </li><li> Para embriões MARCM <ol><li> Coletar embriões para 2h e incubar em um prato de 10 centímetros de Petri rodeada por lenços umedecidos a 25 ° C para uma 2h adicionais. Nota: Ajuste o protocolo de choque térmico altera a freqüência em que clones são gerados. </li><li> Para um número menor de clones, visando única neurônios isolados, submergir e choque térmico em banho-maria por 1h a 38 ° C. </li><li> Para um maior número de clones, de choque térmico para 45mins a 38 ° C, recuperar a RT 30mins, depois de choque térmico novamente para um 30mins adicionais. </li></ol></li><li> Coletar FLP-out embriões para 24h, e de choque térmico por 1h a 38 ° C. </li></ol> 5. Triagem de clones <ol><li> Depois de choque térmico, retire a tampa do arranjo à prova d&#39;água e coloque o suco de maçã placa de ágar numa placa de Petri 10 centímetros cercado por lenços umedecidos. Cultura dos embriões e larvas subseqüentes a 25 ° C até vagaring 3 º instar. Nota: condições de cultura, especialmente nutrição, têm sido mostrados para alterar a morfologia DA arbor dendríticas 32,33. Certifique-se que as larvas crescem têm acesso à pasta de fermento em todos os momentos; monitorar e repor o fermento colar, conforme necessário. </li><li> Deste ponto em diante no protocolo, manipular larvas delicadamente usando uma pinça de insetos. </li><li> Brevemente e lave as larvas na água da torneira, em seguida, coloque em uma placa de ágar. Nota: Este passo reduz de fundo auto-fluorescência de comida ou sujeira na superfície da larva. </li><li> Examine as larvas sob um poderoso microscópio de fluorescência de dissecação. Identificar larvas com GFP-positivos neurônios / células da parede do corpo. </li></ol> 6. Imagem in vivo em de dendritos <ol><li> Coloque a larva em um vidro corrediça depressão com uma pequena gota de glicerol 80%. Coloque uma lamela em slide para imobilizar a larva. Garantir que nenhum ar permanece entre a larva eo coverslip. </li><li> Empurre cuidadosamente a lamínula para rolar a larva para permitir a visualização do neurônio de interesse. A imagem da mCD8:: GFP-etiquetadas arbor dendríticas através de microscopia confocal. </li></ol> 7. Dissecação das larvas Nota antes de começar: dendrites DA neurônio degradam rapidamente após o início da dissecção. Dissecar cada larva individuais em menos de 5min para garantir a morfologia dendrite bom. <ol><li> Dissecar vagando 3 de larvas em uma placa Sylgard como descrito anteriormente 34 com as seguintes modificações: <ol><li> Para a imagem do axônio do neurônio DA termini, certifique-se que o SNC está intacta e os nervos segmentares não são quebradas. Depois de abrir as larvas, primeiro corte cuidadosamente as conexões traqueal contra a parede do corpo e, em seguida, remova cuidadosamente a tripa inteira. </li><li> Se os dendritos de imagem por si só, remova o CNS para permitir um alisador de montagem. </li></ol></li></ol> 8. Fixation e bloqueio de filetes larval <ol><li> Com a larva ainda preso à placa Sylgard, corrigi-lo em PFA 4% em PBS durante 20 minutos à temperatura ambiente em um agitador rotativo delicadamente. </li><li> Remoção de vestígios de tecidos larval (de gordura corporal, traquéia, etc) após a fixação. </li><li> Lavar em PBST (PBS com 0,1% Triton X-100) 10mins 3x na placa Sylgard e shaker. Se examinar termini axônio, manter o filé no prato Sylgard. (Se dendrites coloração é possível transferir os filetes larval a um tubo de 0,5 ml nesta etapa 34) </li><li> Bloquear as larvas de 20mins a RT em 5% de soro normal de burro (NDS) em PBST em um shaker. </li></ol> 9. Coloração dos filetes larval <ol><li> Remover a solução de bloqueio e incubar em anticorpo primário (em 5% NDS / PBST) overnight a 4 ° C em um recipiente Tupperware pequena, rodeada por lenços umedecidos. </li><li> Remover os tubos de 4 ° C e incubar uma hora adicional de RT. </li><li> Lavar 10mins 6x no PBST. Adicione o antib secundárioody (em 5% NDS / PBST), e tampa para evitar a foto-branqueamento fluoróforo 34. </li><li> Incubar quer no RT por duas horas, ou durante a noite a 4 ° C seguido por uma hora em temperatura ambiente. </li><li> Lavar a 10mins larvas 6x no PBST e prossiga para a montagem. </li></ol> 10. Montagem de filetes de larvas para exame do arbor dendrite <ol><li> Monte cada filé larval tão plana quanto possível, utilizando uma tesoura ou um bisturi de dissecação para cortar a cabeça (incluindo os ganchos na boca), ea posterior (incluindo o espiráculos) 34. </li><li> Coloque o filé larval no slide cutícula voltada para baixo, montado em glicerol 80%, e selar os lados da lamela com unha polonês para um &#39;quick&#39; mount 34. </li><li> Para uma montagem permanente e uma imagem mais clara <ol><li> Coloque o filé dissecados larval músculo lateral-down em uma gota de PBS em uma lamela PLL, ele vai rapidamente aderir à lamínula. </li><li> Remover o líquido que for possível após a montagem, however não permitir que ele para secar completamente. </li><li> Pegue a lamela (com filé larval em anexo) através de uma série de etanol: o etanol 35%, seguido por 50%, 70%, 95% e, finalmente, 2x 100% de etanol para cada 10 minutos (o segundo 100% solução EtOH devem ser mudados frequentemente ) e, finalmente, lavar 2-3x 10 minutos em xileno. </li><li> Coloque uma gota de montagem DPX (Distyrene Xileno plastificante) em uma lâmina limpa e cuidadosamente coloque o filé de lamela lado-down no topo. Manter no escuro, e esperar um dia para o DPX para definir antes de imagem. </li></ol></li></ol> 11. Montagem de filetes de larvas para exame dos términos axônio <ol><li> Manter os nervos segmentares correndo entre o PNS e do SNC intacto durante a fixação, a dissecção e coloração para permitir o rastreamento dos axônios das células sensoriais do corpo de neurônio periférico para o VNC. </li><li> Monte cada larval filé cutícula voltada para baixo em 80% de glicerol em um slide depression. Traçar os axônios do corpo celular para o DAVNC sob o microscópio confocal. Nota: PNS neurônios em cada segmento do corpo do projeto de parede para o segmento de cognato do VNC. </li></ol> 12. Resultados representativos: Resultados representativos são mostrados nas figuras 3-5. Fig. 3 mostra a arbor inteiro de um neurônio da classe IV, capturado in vivo sob o microscópio confocal. Fig. 4 mostra um close-up de parte da arbor dendrite de um imunohistoquímica rotulados classe III neurônio que foi corretamente fixada para preservar a morfologia. A inserção associados mostra a degradação que pode ocorrer após uma dissecção sem sucesso e fixação (ambos corados com anti-GFP de anticorpos). Fig. 5 mostra uma única classe IV terminal axônio no SNC (anti-GFP, verde); todos os terminais de classe IV são co-corados com anti-CD2 (magenta). <img alt="Figura 1" src="/files/ftp_upload/3111/3111fig1.jpg" /> Figura 1 Visão geral do protocolo. a) Coletar e, em seguida, de choque térmico-embriões. b)<html> Selecione uma larva com GFP-positivos DA neurônio clone, e depois a imagem do GFP-etiquetadas dendrite arbor na larva viva. c) dissecam a larva, e depois imunohistoquímica manchar o filé. d) Monte o filé manchado, e depois a imagem da árvore dendrítica e projeções axonal do neurônio DA clones. <img alt="Figura 2" src="/files/ftp_upload/3111/3111fig2.jpg" /> Figura 2 Preparação da placa de ágar suco de maçã para o choque térmico. Pegue a placa (seta branca, ab), adicionar uma segunda placa de petri em cima (seta vermelha b) e vedação ao redor com Parafilm (seta azul, b). <img alt="Figura 3" src="/files/ftp_upload/3111/3111fig3.jpg" /> Figura 3 na imagem in vivo do arbor dendrite de um mCD8:: GFP-etiquetadas clone MARCM (genótipo como em 2.1), representando uma classe IV (v&#39;ada) neurônio de uma larva instar 3 rd. Barra de escala é de 50 ìm. d/3111/3111fig4.jpg &quot;/&gt; Figura 4 mCD8:: GFP-etiquetadas MARCM clone de um neurônio III classe (ddaA) corado com anticorpos anti-GFP, mostrando boa morfologia após a dissecção de sucesso e fixação (verde). A inserção mostra a degradação dendrite (setas brancas) que ocorram após dissecção sem sucesso e fixação (magenta). Barra de escala é 25μm. <img alt="Figura 5" src="/files/ftp_upload/3111/3111fig5.jpg" /> Figura 5 O 3 º instar larval VNC. Um clone FLP-out de uma classe VI único terminal axônio (VDAB) (genótipo como em 2.2) é detectada através de anticorpos anti-GFP (verde). Anti-CD2 rótulos toda a classe IV termini (magenta). Barra de escala é 25μm.

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+forward+genetic+screen+for+identifying+mutations+affecting+larval+neuronal+dendrite+development+in+Drosophila+melanogaster.">A novel forward genetic screen for identifying mutations affecting larval neuronal dendrite development in Drosophila melanogaster.</a> Genetics. 172, 2325-2335 (2006).</li><li>Mirouse, V., Swick, L. L., Kazgan, N., St Johnston, D., Brenman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LKB1+and+AMPK+maintain+epithelial+cell+polarity+under+energetic+stress.">LKB1 and AMPK maintain epithelial cell polarity under energetic stress.</a> J Cell Biol. 177, 387-392 (2007).</li><li>Brent, J., Werner, K., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Larval+NMJ+Immunohistochemistry.">Drosophila Larval NMJ Immunohistochemistry.</a> J. Vis. Exp. 25, e1108-e1108 (2009).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Sugimura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+developmental+modes+and+lesion-induced+reactions+of+dendrites+of+two+classes+of+Drosophila+sensory+neurons.">Distinct developmental modes and lesion-induced reactions of dendrites of two classes of Drosophila sensory neurons.</a> J Neurosci. 23, 3752-3760 (2003).</li><li>Zito, K., Parnas, D., Fetter, R. D., Isacoff, E. Y., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Watching+a+synapse+grow:+noninvasive+confocal+imaging+of+synaptic+growth+in+Drosophila.">Watching a synapse grow: noninvasive confocal imaging of synaptic growth in Drosophila.</a> Neuron. 22, 719-729 (1999).</li><li>Landgraf, M., Sanchez-Soriano, N., Technau, G. M., Urban, J., Prokop, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charting+the+Drosophila+neuropile:+a+strategy+for+the+standardised+characterisation+of+genetically+amenable+neurites.">Charting the Drosophila neuropile: a strategy for the standardised characterisation of genetically amenable neurites.</a> Dev Biol. 260, 207-225 (2003).</li></ol>

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A Drosophila larval DA modelo de neurônio fornece um excelente sistema genético para investigar os mecanismos de controle que a morfologia dos neurônios ea formação do circuito. MARCM é geralmente utilizada para rotulagem e para a geração de clones mutantes DA neurônio. Para MARCM que usar uma pan-neural (por exemplo Gal4 C155) ou DA motorista neurônio-específica. Usando um driver de pan-neural é possível usar diretamente diversas unidades populacionais amplamente dispon...

<table border="1"><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comments (opcional) </th></tr><tr><td> SZX16 microscópio de dissecção de fluorescência (com GFPHQ filtro) </td><td> Olimpo </td><td> SZX16 </td><td></td></tr><tr><td> Pinça viver Insect </td><td> FST </td><td> 26030-10 </td><td></td></tr><tr><td> 26 milímetros x 76 milímetros de vidro corrediça depressão </td><td> Toshinriko Co. </td><td> T8-R004 </td><td></td></tr><tr><td> Sylgard 184 (ou Silpot 184) </td><td> Dow Corning </td><td> 3097358-1004 </td><td></td></tr><tr><td> Poli-L-lisina </td><td> Sigma </td><td> P-1524 </td><td> Este produto tem se mostrado mais eficaz </td></tr><tr><td> DPX meio de montagem </td><td> Sigma </td><td> 44581 </td><td></td></tr><tr><td> De coelho anti-GFP </td><td> Invitrogen </td><td> A-11122 </td><td> Diluição 1:500 </td></tr><tr><td> Rat anti-CD8 </td><td> Caltag </td><td> 5H10 </td><td> Diluição 1:200 </td></tr><tr><td> Rato anti-CD2 </td><td> AbD serotec </td><td> MCA443R </td><td> Diluição 1:700 </td></tr><tr><td> Rato anti-Fasciclin2 </td><td> DSHB </td><td> 1d4 </td><td> Diluição de 1:10 </td></tr></table>

morphological analysis of drosophila larval peripheral sensory neuron dendrites and axons using genetic mosaics

Desenvolvimento do sistema nervoso requer a especificação correta de neurônio posição e identidade, seguido por desenvolvimento precisa dendríticas do neurônio específico de classe e fiação axonal. Recentemente, a arborização dendrítica (DA) neurônios sensoriais da larval Drosophila sistema nervoso periférico (SNP) tornaram-se poderosos modelos genéticos em que para elucidar os mecanismos gerais e específicas de classe de diferenciação neuronal. Há quatro principais classes DA neurônio (I-IV) 1. Eles são nomeados em ordem crescente de complexidade arbor dendrite, e têm diferenças de classe específica no controle genético da sua diferenciação 2-10. O sistema DA sensorial é um modelo prático para investigar os mecanismos moleculares por trás do controle da morfologia dendrítica 13/11 porque: 1) pode aproveitar as poderosas ferramentas genéticas disponíveis na mosca da fruta, 2) o neurônio DA dendrite arbor se espalha em apenas duas dimensões sob uma ótica clear cutícula larval tornando-o fácil de visualizar com alta resolução in vivo, 3) a diversidade de classe específico em morfologia dendrítica facilita uma análise comparativa para encontrar elementos-chave controlando a formação de simples contra altamente ramificados árvores dendríticas, e 4) arbor dendríticas estereotipada formas diferentes de neurônios DA facilitar morfométricas análises estatísticas. DA atividade do neurônio modifica a saída de um gerador de padrão larval locomoção centrais 14-16. As diferentes classes de DA neurônio têm distintas modalidades sensoriais, e sua ativação provoca diferentes respostas comportamentais 14,16-20. Além disso diferentes classes de enviar projeções stereotypically axonal no sistema central de Drosophila larval nervoso no cordão nervoso ventral (VNC) 21. Essas projeções terminar com representações topográficas dos dois modalidade neurônio sensorial e DA a posição na parede do corpo do campo dendríticas 7,22De 23. Por isso o exame de projeções DA axonal pode ser usado para elucidar os mecanismos subjacentes mapeamento topográfico 7,22,23, bem como a fiação de um circuito simples locomoção larval modulando 14-17. Nós apresentamos aqui um guia prático para gerar e analisar mosaicos genéticos 24 neurônios marcação DA via MARCM (Análise Mosaic com um marcador de células repressor) 1,10,25 e FLP-out 22,26,27 técnicas (resumidos na Figura 1.).

Watch this Scientific Journal Video about Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics at JoVE.com

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

A arborização dendrítica de neurônios sensoriais da<em&gt; Drosophila</em&gt; Sistema periférico larval nervoso são modelos úteis para elucidar tanto gerais como neurônio específico de classe mecanismos de diferenciação de neurônios. Nós apresentamos um guia prático para gerar e analisar arborização dendrítica mosaicos neurônio genética.

Análise morfológica de Drosophila Larval sensorial periférica Dendrites Neuron e axônios Usando Mosaicos Genéticos

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

morphological-analysis-drosophila-larval-peripheral-sensory-neuron

Research

JoVE Journal

Neuroscience

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

15.2K visualizações.  RIKEN Brain Science Institute. A arborização dendrítica de neurônios sensoriais da Drosophila Sistema periférico larval nervoso são modelos úteis para elucidar tanto gerais como neurônio específico de classe mecanismos de diferenciação de neurônios. Nós apresentamos um guia prático para gerar e analisar arborização dendrítica mosaicos neurônio genética.

Vídeo: Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Em Visualization vivo das vesículas sinápticas Dentro Drosophila larval Axônios segmentar

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Neurociência

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval body wall contains several cell types that are models to study cell and tissue morphogenesis. For example tracheae are used to examine tube morphogenesis1, and the dendritic arborization (DA) sensory neurons of the Drosophila larva have become a primary system for the elucidation of general and neuron-class-specific mechanisms of dendritic differentiation2-5 and degeneration6.
The shape of dendrite branches can vary significantly between neuron classes, and even among different branches of a single neuron7,8. Genetic studies in DA neurons suggest that differential cytoskeletal organization can underlie morphological differences in dendritic branch shape4,9-11. We provide a robust immunological labeling method to assay in vivo microtubule organization in DA sensory neuron dendrite arbor (Figures 1, 2, Movie 1). This protocol illustrates the dissection and immunostaining of first instar larva, a stage when active sensory neuron dendrite outgrowth and branching organization is occurring 12,13.
In addition to staining sensory neurons, this method achieves robust labeling of microtubule organization in muscles (Movies 2, 3), trachea (Figure 3, Movie 3), and other body wall tissues. It is valuable for investigators wishing to analyze microtubule organization in situ in the body wall when investigating mechanisms that control tissue and cell shape.

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how complex cell shapes, such as neuronal dendrites, are achieved during development, it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to examine microtubule organization of dendritic arborization neuron sensory dendrites, trachea, muscle, and other Drosophila larva body wall tissues.

Labeling imuno dos microtúbulos no Dendrites neurônio sensorial, traquéias, e Músculos no Drosophila Wall Corpo Larva

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval ...

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

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

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

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

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

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

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Tensão sensível Gravação Dye de axônios, dendritos e espinhas dendríticas de neurônios individuais em fatias de cérebro

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

Análise Funcional do circuito de alimentação das larvas em Drosophila</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Monte todo imunomarcação de Neurônios Receptores Olfatórios no<em&gt; Drosophila</em&gt; Antena

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

Produção e Isolamento de axônios de neurônios sensoriais para análise bioquímica Usando filtros porosos

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

Remoção de<em&gt; Drosophila</em&gt; Tecido muscular larval Filetes para Análise de imunofluorescência de neurônios sensoriais e células epidérmicas

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Usando Canais Linear de agarose para o Estudo<em&gt; Drosophila</em&gt; Comportamento larval Crawling

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

Análise metabólica do melanogaster da drosófila Larval e adulto cérebros

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...