O projeto descrito foi apoiada pelo Grant Número R01HD050725 do Instituto Nacional de Saúde Infantil e Desenvolvimento Humano para TAG O conteúdo é da exclusiva responsabilidade dos autores e não representam, necessariamente, a posição oficial do Instituto Nacional de Saúde Infantil e Desenvolvimento Humano ou a National Institutes of Health. Agradecemos a membros do laboratório Godenschwege bem como Schreader Barbara para a sua entrada para o manuscrito e vídeo.

1. Dissecção do cordão nervoso ventral Dissecção e corante preenchimento da GF pode ser realizada à temperatura ambiente. Para todas as etapas de dissecção fria (4 ° C) solução salina deve ser usado para desacelerar o metabolismo celular e manter os neurônios vivos por mais tempo. <ol><li> Anestesiar moscas adultas (4 dias ou mais) com CO 2 ou chill-los no gelo até que eles são imobilizados. Anestesiando com gelo vai exigir mais tempo (até 30 min), mas irá manter as moscas imobilizada por mais tempo. </li><li> Coloque uma mosca em uma placa de Petri Sylgard revestido. Esta dissecção requer o uso de uma ampliação alta (80-100X) estereomicroscópio dissecção. </li><li> Com uma tesoura Vannas, remova as pernas, tromba, e asas. </li><li> Use uma pinça grossa e fina pinos de insetos (pinos minutien) para orientar o lado dorsal voar até no prato Sylgard. Pin do abdômen e depois alongar um pouco o animal, colocando dois pinos em cada lado do pescoço, atrás da cabeça, de modo que o pescoço é ligeiramente alongado e acessível (ver figura 2). </li><li> Submergir a voar na nova solução salina Drosophila, frio. Salinas mais comumente usado Drosophila são suficientes para a dissecação, no entanto aqui usamos uma solução salina descrito no Gu e O&#39;Dowd, 2006 4. Uma bolha de ar, cercarão a voar. Retire o ar com uma seringa. </li><li> Faça uma incisão, curto laterais na extremidade dorsal do abdômen. A partir de incisão, faça uma rasa, corte reto no dorso do abdômen para o tórax. Em seguida, continuar o corte raso longitudinal no tórax tão precisamente quanto possível ao longo da linha mediana em direção ao conjuntivo. É fundamental para fazer os cortes rasos para não danificar o VNC (Figura 1). </li><li> Use dois pinos de insetos fina com cuidado abrir o tórax. Espalhe metades do corpo para além usando os pinos e colocar os pinos através dos músculos de vôo (Figura 2). O tórax não deve ser aberta ainda, conforme ilustrado na Figura 2 para evitar rasgar da cutícula. Para garantir uma orientação reta da VNC os pinos devem ser colocados na posição anterior mesmo posterior de cada lado. </li><li> Acesso ao VNC para injeção de corantes e imagem adequada pode ser obstruída por órgãos internos. Portanto, utilize uma pinça e tesouras para remover o estômago, intestino, coração e órgãos reprodutivos. Retirar as glândulas salivares deitado em ambos os lados do VNC. Lavar a dissecção cuidadosa com solução salina fresca para remover o tecido flutuante e gordura. </li><li> Remover a gordura corporal debaixo do conjuntivo cervical (CVC). A gordura corporal é mais visível em uma ampliação alta. Use uma pinça fina para compreender cuidadosamente o tecido do lado e puxe-o para fora. </li></ol> A dissecção pode ser realizada dentro de 5 a 10 minutos. Usando solução salina fria e fresca a dissecados, sistema nervoso intacto permanecerá vivo por pelo menos uma hora. 2. Intracelular GF preencher É recomendado para executar o corante enchimento o mais rapidamente possível após a dissecação. <ol><li> Coloque o prato Sylgard com a mosca dissecadas sob um microscópio vertical com um palco fixo (Figura 3) e um objetivo de ar 10x. Centro da amostra com a extremidade posterior da mosca posicionado em direção ao suporte do eletrodo corante preencher. Use o diferencial de interferência de contraste de modo (DIC) e concentrar-se na CVC. Os axônios grande do FG devem ser claramente visíveis na superfície dorsal da CVC. </li><li> Diluir Lucifer Yellow CH sal dilithium em H2O deionizada a uma concentração de 1%. Puxar um eletrodo de vidro borosilicato com filamento a uma resistência de aprox. 60-80 mohms. A ponta do eletrodo de vidro deve ser longo e afiado. Encha a ponta do eletrodo com 1% de solução Lucifer Yellow colocando o eletrodo com a extremidade oposta a ponta do eletrodo na solução Lucifer Yellow por aprox. 30-60 segundos. Em seguida, o eletrodo de aterramento com LiCl 3M, utilizando uma seringa Hamilton deixando uma bolha de ar entre o corante e LiCl. Conecte o eletrodo corante de preenchimento para o palco cabeça. </li><li> Inserir um cloreto de prata revestido eletrodo terra fio no soro fisiológico e colocar o eletrodo corante encher acima do paralelo voar para o VNC usando um micromanipulador 3D hidráulico. O eletrodo de corante de preenchimento deve ser conectado a um amplificador que permite a passagem de corrente a fim de liberar negativa ou positivamente carregada corantes do eletrodo, passando hiper ou despolarizantes atual, respectivamente. </li><li> Mudar para uma lente 40x de imersão em água e menor a ponta do eletrodo corante preencher em uma das GFs. Os axônios GF ficava perto da superfície dorsal da CVC. Use um movimento (em direção à cabeça) para a frente para inserir o eléctrodo na GF. </li><li> Alterar a sua fonte de luz para iluminar epifluorescência e mudar para um filtro de Lucifer Yellow. Aplicar uma corrente hiperpolarizante ao eletrodo corante preencher com o amplificador para injetar o Yellow Lucifer no GF. A quantidade de corante injetado pode ser controlada por diaquantidade de corrente e passados, bem como a duração da corrente é passado. Para evitar que a tinta se espalhe para fora do local da injeção ou danificar o GF, recomenda-se injetar com uma corrente baixa (cerca de 3-5 nA) mais 1-5 minutos. </li><li> Aplicar atual até o terminal GF na VNC pode ser visto claramente (Figura 4A). Para trans-sináptica dos neurônios pós-sinápticos coloração, continuar a passar corrente em até dye também pode ser visto nos neurônios pós-sinápticos. Desligue a corrente e retire o eletrodo corante preencher. </li></ol> 3. Resultados representativos: Um corante representante de preenchimento para uma GF com Lucifer Yellow é mostrado na Figura 4A. A imagem foi adquirida como uma pilha de imagens no software Nikon Elements usando uma câmera montada no local no microscópio. Neste atual amostra foi passada através do eletrodo por cerca de 2 minutos. O GF é completamente preenchido eo terminal GF grande no tórax é bem marcada (Figura 4A, elipse). Não neurônios pós-sinápticos são visíveis neste caso, embora Lucifer Yellow é suficientemente pequeno para passar através junções. Se o corante não é injetado o tempo suficiente para a GF, em seguida trans-sináptica enche de neurônios pós-sinápticos podem não foi visto porque o sinal é muito fraco para ser detectado. Neurônios pós-sinápticos para o FG (Figura 4B, cabeças de seta) podem ser visualizados de forma mais confiável usando ainda menor traçadores neuronais, como Neurobiotin. Injeção de um Neurobiotin 7% em dois solução de acetato de potássio M com uma corrente despolarizante por 2 minutos é suficiente para rotular neurônios pós-sinápticos fortemente. Neurobiotin podem ser visualizados por microscopia confocal usando Estreptavidina acoplados a corantes de diferentes comprimentos de onda. Neurobiotin porque não é um corante fluorescente, co-injeção de Dextran ou Rhodamin-hidrazonas Alexa na mesma solução, pode ser usado para monitorar uma injeção de sucesso para o GF (Figura 4C, de Boerner e Godenschwege, 2010 5). <img src="/files/ftp_upload/3080/3080fig1.jpg" alt="Figura 1" /> Figura 1. Desenho esquemático de uma mosca visto lateralmente. O tórax é exibido com uma incisão longitudinal de visualizar as estruturas internas. Durante a dissecção, a dorsal músculos de vôo longitudinal (DLMS, azul) foram cortadas, sem danificar o cordão nervoso ventral (VNC, vermelho escuro), que fica ventral aos músculos de vôo. O intestino (verde) está acima do VNC. <img src="/files/ftp_upload/3080/3080fig2.jpg" alt="Figura 2" /> Figura 2. Vista dorsal de uma mosca dissecados. Dois pinos são colocados atrás da cabeça para posicionar a voar. Os pinos afixada através dos músculos de vôo (DLM) mantenha o tórax aberto. Órgãos internos foram removidos para expor o cordão nervoso ventral (VNC). <img src="/files/ftp_upload/3080/3080fig3.jpg" alt="Figura 3" /> Figura 3. Dye configuração de preenchimento. <img src="/files/ftp_upload/3080/3080fig4.jpg" alt="Figura 4" /> Figura 4. A) Z-projeção da parte posterior da VNC mostrando uma GF preenchido com Lucifer Yellow. A imagem foi adquirida imediatamente após o preenchimento da tintura com uma câmera no local. No tipo selvagem animais, o terminal (elipse) no tórax é claramente visível. B) imagem confocal de um corante GF preencher com Neurobiotin em uma mosca do tipo selvagem. Ambos os GFs (setas) e neurônios pós-sinápticos para o FG (seta cabeças) são rotulados. Neurobiotin foi visualizado com Estreptavidina acoplado a Cy3. C) imagem confocal de tipo selvagem FG preenchido com Alexa555.

<ol>
	<li>Power, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thoracico-abdominal+nervous+system+of+an+adult+insect+Drosophila+melanogaster.">The thoracico-abdominal nervous system of an adult insect Drosophila melanogaster.</a> J. Comp. Neurol. 88, 347-409 (1948).</li><li>Allen, M. J., Godenschwege, T. A., Tanouye, T. A., Phelan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+an+escape:+development+and+function+of+the+Drosophila+giant+fibre+system.">Making an escape: development and function of the Drosophila giant fibre system.</a> Semin. Cell Dev. Biol. 17, 31-41 (2006).</li><li>Boerner, J., Duch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Average+shape+standard+atlas+for+the+adult+Drosophila+ventral+nerve+cord.">Average shape standard atlas for the adult Drosophila ventral nerve cord.</a> J. Comp. Neurol. 518, 2437-2455 (2010).</li><li>Gu, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+synaptic+transmission+in+adult+Drosophila+Kenyon+cells+in+sity.">Cholinergic synaptic transmission in adult Drosophila Kenyon cells in sity.</a> J. Neurosci. 26, 265-272 (2006).</li><li>Boerner, J., Godenschwege, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+for+the+Drosophila+ventral+nerve+cord+standard+in+neuronal+circuit+reconstruction+and+in-depth+analysis+of+mutant+morphology.">Application for the Drosophila ventral nerve cord standard in neuronal circuit reconstruction and in-depth analysis of mutant morphology.</a> J. Neurogent. 24, 158-1567 (2010).</li><li>Godenschwege, T. A., Kristiansen, L. V., Uthaman, S. B., Hortsch, M., Murphey, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conderved+role+for+Drosophila+Neuroglian+and+human+L1-CAM+in+central-synapse+formation.">A conderved role for Drosophila Neuroglian and human L1-CAM in central-synapse formation.</a> Curr. Biol. 16, 12-23 (2006).</li><li>Uthaman, S. B., Godenschwege, T. A., Murphey, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanism+distinct+from+highwire+for+the+Drosophila+ubiquitin+conjugase+bendless+in+synaptic+growth+and+maturation.">A mechanism distinct from highwire for the Drosophila ubiquitin conjugase bendless in synaptic growth and maturation.</a> J. Neurosci. 28, 8615-8623 (2008).</li><li>Storkebaum, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dominant+mutations+in+the+tyrosyl-tRNA+synthetase+gene+recapitulate+in+Drosophila+features+of+human+Charcot-Marie-Tooth+neuropathy.">Dominant mutations in the tyrosyl-tRNA synthetase gene recapitulate in Drosophila features of human Charcot-Marie-Tooth neuropathy.</a> Proc. Natl. Acad. Sci. USA. 106, 11782-11787 (2009).</li><li>Allen, M. J., Godenschwege, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+recordings+from+the+Drosophila+giant+fiber+system+(GFS).">Electrophysiological recordings from the Drosophila giant fiber system (GFS).</a> Cold Spring Harb. Protoc. 2010, pdb.prot5453-pdb.prot5453 (2010).</li><li>Augustin, H., Allen, M. J., Partridge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+Recordings+from+the+Giant+Fiber+Pathway+of+D.+melanogaster.">Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster.</a> J. Vis. Exp. , (2011).</li></ol>

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O sistema nervoso pode ser dissecada sem extraí-lo do corpo da mosca. Isso tem duas vantagens, primeiro, a dissecção causa pouco dano ao sistema nervoso e, segundo, a maioria dos nervos ficar ligado para os músculos e órgãos sensoriais. Realizar a dissecção, como descrito, prepara a amostra para a frente de corante rotulagem do FG. Convenientemente, os motoneurônios pós-sináptico para o FG ficar ligado para os músculos. Axônios, portanto, não estão danificados, mantendo os neurônios vivos por mais tempo....

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> Sylgard 184 Kit de elastômero de silicone </td><td> Dow Corning </td><td></td><td></td></tr><tr><td> Microscópio dissecção </td><td> AmScope </td><td> SM-2TZ </td><td></td></tr><tr><td> Vannas Superfine Tesoura </td><td> Instrumentos acadêmica </td><td> VS1023 </td><td></td></tr><tr><td> Pinça Dumont Dumontstar 55 </td><td> Multa Ferramentas Ciência </td><td> 11295-51 </td><td></td></tr><tr><td> Insect pinos Austerlitz </td><td> Multa Ferramentas Ciência </td><td> 26002-10 </td><td> Ø 0,1 milímetros </td></tr><tr><td> Eletrodos de vidro de borosilicato </td><td> Precision Instruments mundo </td><td> 1B100F-4 </td><td></td></tr><tr><td> Vertical Pipetar Puller 700c </td><td> David Kopf Instruments </td><td> Modelo 700C </td><td></td></tr><tr><td> Lucifer Yellow CH dilithium sal </td><td> Sigma </td><td> L0259 </td><td> 1% em H 2 O </td></tr><tr><td> Microscópio Stage fixo Upright &amp; Camera </td><td> Nikon </td><td> FN-1 e DS-U2 Câmera </td><td></td></tr><tr><td> Plano de Fluor Objetivo Air 10X </td><td> Nikon </td><td> CFI Plano Fluor 10X NA 0,3 WD 16 milímetros </td><td></td></tr><tr><td> Fluor água 40X mergulhando Objetivo </td><td> Nikon </td><td> CFI Fluor 40X W NA 0,80 WD 2,0 milímetros </td><td></td></tr><tr><td> Micromanipulador hidráulico 3D </td><td> Narishige Internacional </td><td> Modelo MW0-3 </td><td></td></tr><tr><td> Amplificador </td><td> Ficando Instruments, Inc. </td><td> 5A modelo </td><td></td></tr></tbody></table>

whole mount preparation of the adult drosophila ventral nerve cord for giant fiber dye injection

Para analisar a morfologia axonal e dendríticas dos neurônios, é essencial para obter a rotulagem exacta de estruturas neuronais. Preparação de amostras bem rotulados com pouco ou nenhum dano tecidual nos permite analisar a morfologia das células e comparar amostras individuais entre si, permitindo assim a identificação de anomalias mutantes. No método de dissecação demonstrou o sistema nervoso permanece na maior parte dentro da mosca adulta. Através de uma incisão dorsal, abdome e tórax são abertas ea maioria dos órgãos internos são removidos. Apenas o lado dorsal da medula nervosa ventral (VNC) e do conjuntivo cervical (CVC), que contém os axônios das fibras grandes gigantes (FG) 1 estão expostos, enquanto o cérebro com o corpo celular e dendritos GF 2 permanece na cabeça intacta . Nesta preparação mais nervos do VNC deve permanecer solidários com os seus músculos. Após a dissecção, o preenchimento intracelular da fibra gigante (GF) com um corante fluorescente é demonstrada. Na CVC os axônios GF estão localizados na superfície dorsal e, portanto, podem ser facilmente visualizadas em um microscópio com contraste de interferência diferencial (DIC) óptica. Isto permite a injeção de axônios GF com corante neste site para rotular o GF inteiro, incluindo os axônios e os terminais na VNC. Este método resulta em coloração confiável e forte da FG permitindo que os neurônios a ser trabalhada imediatamente após o enchimento com um microscópio de epifluorescência. Alternativamente, o sinal fluorescente pode ser melhorada através de procedimentos imuno-histoquímica padrão de 3 adequado para microscopia confocal de alta resolução.

Watch this Scientific Journal Video about Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection at JoVE.com

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

Um<em&gt; In vivo</em&gt; Dissecção do adulto<em&gt; Drosophila</em&gt; Cordão nervoso ventral (VNC) é demonstrada. Este método de dissecação em particular cause poucos danos ao VNC permitindo a rotulagem subseqüentes dos neurônios fibra gigante com tinta fluorescente para imagens em alta resolução.

Preparação Monte todo o Adulto Drosophila Cordão nervoso ventral para injeção gigante de fibra Dye

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

whole-mount-preparation-adult-drosophila-ventral-nerve-cord-for-giant

Research

JoVE Journal

Neuroscience

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

19.0K visualizações.  Florida Atlantic University. Um In vivo Dissecção do adulto Drosophila Cordão nervoso ventral (VNC) é demonstrada. Este método de dissecação em particular cause poucos danos ao VNC permitindo a rotulagem subseqüentes dos neurônios fibra gigante com tinta fluorescente para imagens em alta resolução.

Vídeo: Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

Visualização do Sistema Nervoso embrionárias no Whole-mount Drosophila Os embriões

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Neurociência

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

As gravações eletrofisiológicas da via fibra Gigante da D. melanogaster</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

Nanoinjection emparelhado e Eletrofisiologia Ensaio para a tela de bioatividade dos compostos utilizando os Drosophila melanogaster Sistema Fibra Gigante

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Reprodutível mouse esmagamento do nervo ciático e subsequente avaliação de Regeneração por Análise Whole Mount muscular

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

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 ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

Dissecção do Músculo Transversus Abdominis para Análise de Junção Neuromuscular de Montagem Integral

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

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 ...

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional afferent fibers monitor other characteristics of the muscle environment, including metabolite buildup, temperature, and nociceptive stimuli. Overall, abnormal activation of sensory neurons can lead to movement disorders or chronic pain syndromes. We describe the isolation of the extensor digitorum longus (EDL) muscle and nerve for in vitro study of stretch-evoked afferent responses in the adult mouse. Sensory activity is recorded from the nerve with a suction electrode and individual afferents can be analyzed using spike sorting software. In vitro preparations allow for well controlled studies on sensory afferents without the potential confounds of anesthesia or altered muscle perfusion. Here we describe a protocol to identify and test the response of muscle spindle afferents to stretch. Importantly, this preparation also supports the study of other subtypes of muscle afferents, response properties following drug application and the incorporation of powerful genetic approaches and disease models in mice.

An In Vitro Adult Mouse Muscle-nerve Preparation for Studying the Firing Properties of Muscle Afferents

Muscle sensory neurons are involved in proprioceptor signaling and also report on metabolic state and injury related events. We describe an adult mouse in vitro muscle-nerve preparation for studies on stretch-activated muscle afferents.

Um<em&gt; In Vitro</emPreparação Muscle-nervo&gt; Adulto mouse para estudar as propriedades de queima de músculos aferentes

Muscle sensory neurons innervating muscle spindles and Golgi tendon organs encode length and force changes essential to proprioception. Additional ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

Monte todo Dissecção e Imunofluorescência do mouse cóclea Adulto

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

A Preparação de Oblique Spinal Cord Fatias de Estimulação raiz ventral

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

Preparação Monte todo o Adulto Drosophila Cordão nervoso ventral para injeção gigante de fibra Dye

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