Com a subvenção de NIH R01 MH 099557

1. fabricação de eletrodos de gravação <ol><li>puxando os eletrodos de vidro <ol><li>uso o seguinte protocolo para o extrator de microeletrodos (ver Tabela de materiais): <ol><li>linha 1: calor 510 puxar - velocidade 30 hora 250; Linha 2: Aqueça 490 Pull - velocidade 30 hora 250. 
			Nota: As unidades de tempo correspondem a 0,5 ms por unidade; as outras unidades são relativas. O valor do calor deve ser ajustado para cada filamento após o teste de rampa é executado.</li>
		</ol></li> <li>Usar um microscópio (ampliação de x 35) para garantir que o diâmetro interno do eletrodo puxado é na faixa de 7 a 10 µm ( figura 1A). Armazenar os capilares em recipientes hermeticamente fechados para evitar a acumulação de poeira.</li>
	</ol></li> <li>Fogo polimento <ol><li>capilares de polonês de fogo (80-90% do valor máximo de calor para 1-2 s) usando uma microforja (ver Tabela de materiais). Certifique-se o diâmetro interno final do eletrodo polido é de 5 µm ( figura 1A) 27.</li>
	</ol></li> <li>Dobra Nota: duas curvas são feitas a fim de posicionar os eletrodos na parte superior do músculo sob um objectivo de alta ampliação.
	<ol><li>Uso o aparelho mostrado na figura 1B. Fixar o eletrodo no manipulador e posicione a ponta sobre o filamento, não tocando la</li>
		<li>Conjunto valor para 60-70% do máximo de calor e pressione o pedal da microforja para 1-2 s (consulte a Tabela de materiais) para aquecer o filamento. Use uma agulha em forma de L para puxar suavemente a ponta do eletrodo ( figura 1B alargada). Faça a curva em cerca de 90 o ( Figura 1).</li>
		<li>Segure o eletrodo sobre a chama da tocha à mão usando a pinça e fazer a segunda curva a uma distância de 7-10 mm da primeira curva e em um ângulo de aproximadamente 120 ó ( Figura 1).</li>
	</ol></li></ol> <img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56493/56493fig1.jpg" /> Figura 1. Passos finais da fabricação de micropipeta. (A) eletrodo dicas depois puxando e polimento de fogo. (B. 1) a instalação para a ponta dobra. (B. 2), a área de Box é alargada mostrado à direita. O eletrodo e o filamento são fixos para que a ponta do eletrodo é posicionado ligeiramente acima do fio e não tocar. (C. 1) o eletrodo de gravação. (C. 2), a área de Box é alargada mostrado à direita. A primeira curva tem um ângulo de aproximadamente 90°, e a distância entre a primeira curva e a ponta do eletrodo é de cerca de 1 mm. escala barra = 3 mm. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig1large.jpg" target="_blank"> Clique aqui para ver uma versão maior desta figura.</a>
 2. etapas preparatórias adicionais <ol><li>Prepare-se, como heamolymph (HL3) solução (em mM): NaCl 70, 5 KCl, 20 MgCl 2, 10 NaHCO 3, 5 a trealose, 115 sacarose, 5 HEPES e 1mm CaCl 2; ajustar o pH para 7,3 - 7.4. manter a solução na geladeira e torná-lo fresco toda semana.</li>
	<li>Fazer o mesmo caminho como gravação de vidro de pipetas de estimulação pipetas, exceto que não há nenhuma necessidade de dobrá-los. 
	Nota: A preparação de eletrodos de estimulação é descrita detalhadamente em 28 e 27. O diâmetro final após polimento de fogo deve ser na faixa de 5-7 µm.</li>
	<li>Inserir uma pipeta de estimulação em um suporte de microeletrodos conectada a uma seringa.</li>
	<li>Placas de dissecação de fabrico de pequenos pratos de petri (35 x 10mm) revestidos com epóxi de borracha de silicone (consulte Tabela de materiais), conforme descrito em 28. 
	Nota: A borracha de Silicone deve ser completamente endurecida antes do uso.</li>
	<li>Escolher um errante larvas de terceiro instar e dissecá-lo conforme descrito em 27 , 28 , 29 , 30. 
	Nota: Para visualizar boutons, use a estirpe voar CD8-GFP (ver Tabela de materiais) <ol><li>usando fórceps (ver Tabela de materiais), escolher as 3 rd ínstar larvas de um frasco.</li>
		<li>Pin as larvas, colocando o primeiro pino posterior e outro pino anterior (fechar a boca hooks).</li>
		<li>Solução Adicionar HL3.</li>
		<li>Fazer um corte usando Primavera tesoura (ver Tabela de materiais) todo o caminho do pino de topo para o fundo um do lado dorsal das larvas.</li>
		<li>Pino o filete de larvas, colocação de 2 pinos adicionais sobre os lados esquerdo e direito das larvas.</li>
		<li>Remover a coragem e a tracheas usando fórceps.</li>
		<li>Cortar os nervos fora o cabo ventral do nervo usando tesouras primavera.</li>
	</ol></li></ol> 3. Gravações elétricas de EJCs <img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56493/56493fig2.jpg" /> Figura 2. A configuração de gravação. A amostra MNJ derrotou o silicone revestido de Petri (seta) é posicionado sobre o estágio móvel do vertical composto microscópio equipado com recursos de epifluorescência, um objectivo de alta ampliação e dois Micromanipuladores. O microscópio está estacionado em uma tabela de antivibração. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig2large.jpg" target="_blank"> clique aqui para ver uma versão maior desta figura.</a>
<ol><li>Gravação <ol><li>lugar de Petri com a preparação na fase de microscópio ( Figura 2). Inserir o eléctrodo de referência no banho.</li>
		<li>Preencher o eletrodo gravação com HL3 solução. No âmbito do objectivo de 10x (consulte Tabela de materiais), mergulhe o eletrodo para o banho e coloque-o sobre os músculos, 6 e 7 dos segmentos abdominais 2, 3 ou 4 usando o micromanipulador ((ver Tabela de materiais) Figura 3 A. 1 e a. 2).</li>
		<li>Mudar o objetivo para 60 x (veja a Tabela de materiais). Focar a área de interesse usando epi-fluorescência ou óptica DIC. Colocar a ponta do eletrodo em cima bouton synaptic ( Figura 3 b. 1-3).</li>
		<li>Pressione o eletrodo muito suavemente para o músculo. A pressão excessiva pode danificar o MNJ ou induzir um aumento na atividade sináptica espontânea. Verifique se a ponta do eletrodo não está entupido - se substituí-lo, é</li>
		<li>Ligar o amplificador, placa A/D e o computador.</li>
		<li>Escolher o modo de grampo de tensão do amplificador.</li>
		<li>Iniciar o software de aquisição e escolha o &#39; sem lacunas &#39; modo.</li>
		<li>Observe a aparência do mEJCs na tela do computador.</li>
		<li>Assegure-se que a amplitude do mEJCs é na faixa de 0,2 - 0,7 nd. 
		Nota: EJCs menores indicam que o eléctrodo de gravação tem defeitos ou se ele não está posicionado corretamente.</li>
	</ol></li></ol> <img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56493/56493fig3.jpg" /> Figura 3. Visualização de boutons sinápticas. (A), A imagem de brightfield de um hemi-segmento abaixo dos 10 x ampliação (a. 1) e a área ampliada Box mostrando os músculos 6 e 7 (a. 2, a seta marca o eléctrodo de gravação). Barra de escala = 50 µm. (B) sináptica boutons são visualizados em uma linha de drosófila com um marcador neuronal geneticamente codificado (CD8-GFP) usando imagens de epi-fluorescência (b. 1) ou óptica DIC (b. 2). Imagens são tiradas com o 60 x objetivo e o cubo do filtro para a imagem latente de GFP (ver Tabela de materiais). Synaptic boutons são marcados com as setas e uma sobreposição de imagens DIC e fluorescente é mostrada em b. 3. Barra de escala = 10 µm. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig3large.jpg" target="_blank"> clique aqui para ver uma versão maior desta figura.</a>
<ol start="2"> <li>estimulação <ol><li>usando um micromanipulador, sob um controle visual, coloque o eletrodo de estimulação perto do axônio que inervam segmentos abdominais 2-4.</li>
		<li>Aplicar pressão negativa puxando o êmbolo da seringa conectada ao titular do eletrodo, para que o axônio é puxado para dentro do eletrodo ( Figura 2).</li>
		<li>Ativar o estimulador. Gire o botão na unidade de isolamento (consulte a Tabela de materiais) para definir uma corrente zero e suavemente, aumente até EJCs aparecem (ou até que o limite é atingido).</li>
		<li>Realizar a estimulação em um regime de suprathreshold, com a estimulação atual aumentou aproximadamente duas vezes, em comparação com o limiar para a observação de EJCs. 
		Nota: Em nossa experiência, tal intensidade de estimulação é ideal para evitar falhas de potencial de ação e potencial de ação disparando. Por exemplo, se EJCs aparecem na corrente de estimulação de 0.2 mA, usar uma corrente de 0,4 mA durante todo o experimento.</li>
	</ol></li> <li>Selo resistência <ol><li>medir a resistência de selo do eletrodo gravação macropatch colocando o interruptor de resistência do eléctrodo do amplificador para &#34; teste do selo &#34; posição. Observe que o valor da resistência do selo em GΩ será exibido no &#34; atual &#34; janela.</li>
		Certifique-se da resistência do selo está no intervalo de 0,5 - 2 M <li>&#8486;. Valores fora desse intervalo indicam que o eletrodo é polido não corretamente ou não devidamente posicionado.</li>
		<li>Monitorar a resistência do selo em toda a gravação, certificando-se que permanece constante durante todo o experimento.</li>
	</ol></li></ol> 4. Análise <ol><li>analisar gravações empregando personalizado software para análise (ver Tabela de materiais). 
	Nota: Nosso laboratório utiliza software in-house Quantan 31, que é personalizado para a detecção de EJCs e mEJCs gravado focally ( Figura 4). Este software inclui um filtro Gaussian digital e permite a detecção de picos quantal em sobreposição multi quantal eventos ( Figura 4A). Outras abordagens são descritas em 17.</li>
</ol> <img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56493/56493fig4.jpg" /> Figura 4. Análise de quantal. Deteção de mEJCs (A) e (B) de EJCs por Quantan software. A área do evento é marcada em verde, e picos são marcados por setas vermelhas. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig4large.jpg" target="_blank"> clique aqui para ver uma versão maior desta figura.</a>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+synaptic+plasticity+in+Drosophila+memory+mutants+with+a+defective+cyclic+AMP+cascade.">Altered synaptic plasticity in Drosophila memory mutants with a defective cyclic AMP cascade.</a> Science. 251 (4990), 198-201 (1991).</li><li>Delgado, R., Maureira, C., Oliva, C., Kidokoro, Y., Labarca, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+of+vesicle+pools,+rates+of+mobilization,+and+recycling+at+neuromuscular+synapses+of+a+Drosophila+mutant,+shibire.">Size of vesicle pools, rates of mobilization, and recycling at neuromuscular synapses of a Drosophila mutant, shibire.</a> Neuron. 28 (3), 941-953 (2000).</li><li>Melom, J. E., Akbergenova, Y., Gavornik, J. P., Littleton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+and+evoked+release+are+independently+regulated+at+individual+active+zones.">Spontaneous and evoked release are independently regulated at individual active zones.</a> J Neurosci. 33 (44), 17253-17263 (2013).</li></ol>

Os autores não têm nada para divulgar.

Drosófila representa um organismo modelo vantajoso para estudar a transmissão sináptica. Várias configurações de gravação foram usadas no MNJ larval, incluindo gravações intracelulares de potenciais sinápticos, gravações das correntes sinápticas com dois eletrodos tensão braçadeira33,34e macropatch focal gravações das correntes sinápticas descritas aqui. A última técnica permite a quantificação precisa da transmissão sináptica em...

Drosófila é um sistema excelente modelo para estudar os mecanismos moleculares de controle da transmissão sináptica. O sistema neuromuscular em Drosophila é glutamatérgico, e portanto a drosófila de junção neuromuscular (JNM) pode ser usada para estudar as características conservadas de lançamento glutamatérgico. Desde Jan e de Jan estudo1, as terceiro ínstar larvas tem sido amplamente costumava estudar evocado e espontânea transmissão sináptica monitorando potenciais de junção excitatória (EJPs) ou correntes (EJCs). EJPs são comumente registrados intracelular com um microeléctrodo de vidro afiado, e refletem a atividade do MNJ inteira, incluindo todos os boutons fazendo sinapses para a fibra muscular determinada.
Em contraste, a atividade de um número limitado dos sítios de lançamento pode ser gravada focally posicionando uma dica micropipeta perto de terminais neuronais ou varicosidades sinápticas. Esta técnica foi originalmente empregada por Katz e Miledi2, e gravações extracelulares focais têm sido empregadas com sucesso em várias preparações de JNM, incluindo sapo3,4,5, rato6 , 7 , 8, crustáceo,9,10,11,12,13,14,15,16e Drosófila17,18,19,20,21,22,23. Esta abordagem foi desenvolvida por Dudel, quem otimizado macropatch recodificação eletrodos24,25. Na implementação do Dudel, esta técnica estreitamente alinhados a solta-remendo-braçadeira método26.
O MNJ larvas de Drosophila definiu claramente boutons sinápticas e linhas transgénicas com geneticamente codificadas etiquetas fluorescentes neuronais (ver Tabela de materiais) estão prontamente disponíveis. Estas vantagens nos permitiram gravar EJCs e mEJCs de um selecionado bouton sináptica20,21,22. Aqui, descrevemos esta técnica em detalhe.

<table><tbody><tr><td>Sutter P-97</td><td>Sutter instrument</td><td>P-97</td><td>Microelectrode puller</td></tr><tr><td>Narishige MF-830</td><td>Narishige</td><td>MF-830</td><td>Microforge</td></tr><tr><td>WPI MF200</td><td>WPI</td><td>MF200</td><td>Microforge</td></tr><tr><td>Glass capilaries</td><td>WPI</td><td>B150-86-10</td><td>Glass capilaries</td></tr><tr><td>Microtorch 1WG61</td><td>Grainer</td><td>1WG61</td><td>Microtorch</td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td>SYLGARD 184</td><td>Silicone for dissection plates preparation</td></tr><tr><td>Dissection pins</td><td>Amazon</td><td>B00J5PMPJA</td><td>Pins for larvae positioning</td></tr><tr><td>Tweezers</td><td>WPIINC</td><td>500342</td><td>Tweezers for placing pins, removing the guts and tracheas.</td></tr><tr><td>Scissors</td><td>WPIINC</td><td>501778</td><td>Scissors for cutting the cuticula of the larvae and nerves.</td></tr><tr><td>Olympus BX61WI</td><td>Olympus</td><td>BX61WI</td><td>Upright microscope</td></tr><tr><td>Olympus Lumplan FL N 60x</td><td>Olympus</td><td>UPLFLN 60X</td><td>Microscope objective 60X</td></tr><tr><td>Olympus UPlan FL N 10x</td><td>Olympus</td><td>Uplanfl N 10X</td><td>Microscope objective 10X</td></tr><tr><td>Narishige Micromanipulator</td><td>Narishige</td><td>MHW-3</td><td>Three-axis Water Hydraulic Micromanipulator</td></tr><tr><td>npi Electronic GmbH ELC-03XS</td><td>npi Electronic GmbH</td><td>ELC-03XS</td><td>Electrophysiological amplifier</td></tr><tr><td>A.M.P.I Master 8</td><td>A.M.P.I.</td><td>Master 8</td><td>Electrical stimulator</td></tr><tr><td>A.M.P.I Iso-Flex</td><td>A.M.P.I.</td><td>Iso-Flex</td><td>Stimulus isolator</td></tr><tr><td>TMC antivibration table</td><td>TMC</td><td>63-9090</td><td>Antivibration table</td></tr><tr><td>TMC Faraday cage</td><td>TMC</td><td>81-333-90</td><td>Faraday cage</td></tr><tr><td>Digidata 1322A</td><td>Axon Instruments</td><td>Digidata 1322A</td><td>Digidata</td></tr><tr><td>Computer</td><td>Dell</td><td>Dell Dimension 5150</td><td>Computer with Win XP OS</td></tr><tr><td>Electrode holder</td><td>WPI</td><td>MEH3SW</td><td>Electrode holder</td></tr><tr><td>Optical filter</td><td>Omega optical</td><td>XF 115-2</td><td>Filter cube for Green Fluorescent Protein (GFP) detection</td></tr><tr><td>pCLAMP 8</td><td>Axon Instruments</td><td>8.0.0.81</td><td>Software for signal recording</td></tr><tr><td>Quantan</td><td>In-house software</td><td>-</td><td>Software for signal processing</td></tr><tr><td>Canton-S (Wildtype)</td><td>Bloomington Stock Center</td><td>64349</td><td>Control fly line</td></tr><tr><td>cpx SH1</td><td>Generous Gift of J.T. Littleton</td><td>-</td><td>Complexin knock-out fly line with increased spontaneous exocytosis</td></tr><tr><td>CD8-GFP</td><td>Bloomington Stock Center</td><td>5137</td><td>Fly line with neuronal fluorescent (GFP) Tag</td></tr></tbody></table>

focal macropatch recordings of synaptic currents from the drosophila larval neuromuscular junction

Drosófila junção neuromuscular (JNM) é um sistema excelente modelo para estudar a transmissão sináptica glutamatérgico. Descrevemos a técnica de macropatch focal gravações das correntes sinápticas de boutons visualizados no MNJ larvas de Drosophila . Esta técnica requer a fabricação personalizada de micropipetas, bem como um microscópio composto, equipado com uma alta ampliação, objectivo de imersão de água de longa distância, óptica de interferência diferencial (DIC) de contraste e uma fluorescente de gravação acessório. O eletrodo de gravação está posicionado no topo de um selecionado bouton sináptica visualizado com óptica DIC, epi-fluorescência ou ambos. A vantagem desta técnica é que permite monitorar a atividade sináptica de um número limitado de sítios de lançamento. O eletrodo de gravação tem um diâmetro de vários micra, e os locais de lançamento posicionados fora da borda do eletrodo não afetam significativamente as correntes gravadas. As correntes sinápticas gravadas tem cinética rápida e podem ser facilmente resolvidas. Estas vantagens são especialmente importantes para os estudos de linhas de mosca mutantes com maior atividade sináptica espontânea ou assíncrona.

Macropatch focal gravações permitem monitoramento atividade sináptica de boutons sinápticas selecionados (Figura 5). Quando o eletrodo é posicionado na parte superior uma bouton synaptic (Figura 5A, site 1), os mEJCs gravados (Figura 5, local 1) têm uma amplitudes exceda significativamente o nível de ruído e em ponto de aumentação fases (em uma escala do milissegundo sub). Quando o eletrodo...

Watch this Scientific Journal Video about Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction at JoVE.com

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Macropatch focal gravações das correntes sinápticas da junção Neuromuscular Larval da drosófila

Correntes sinápticas podem ser gravadas focally de boutons sinápticas visualizados na drosófila terceiro ínstar larvas junção neuromuscular. Esta técnica permite monitorar a atividade de um único bouton sináptica.

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

focal-macropatch-recordings-synaptic-currents-from-drosophila-larval

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

6.0K visualizações.  Wayne State University. Correntes sinápticas podem ser gravadas focally de boutons sinápticas visualizados na drosófila terceiro ínstar larvas junção neuromuscular. Esta técnica permite monitorar a atividade de um único bouton sináptica.

Vídeo: Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

Gravações patch emparelhado Grampo da Motor-neurônio e músculo alvo ósseas em Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

Neurociência

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

Dissecção e imagem das Zonas de Ativos no Drosophila Junção neuromuscular

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

De gravação simultânea de sinais de cálcio dos neurônios identificados e comportamento alimentar de Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

Métodos eletrofisiológicos de gravação Potenciais Synaptic do MNJ de larvas de Drosophila

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Biologia

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited.

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.

Channelrhodopsin2 Estimulação Mediated de Potenciais Synaptic em Drosophila Junções Neuromusculares

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available ...

Preparation of Drosophila Central Neurons for in situ Patch Clamping

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker1,2. Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain3,4 and ventral nerve cord of embryonic5,6, larval7,8,9,10, and adult Drosophila11,12,13,14. A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN515), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.

Preparation of Drosophila Central Neurons for in situ Patch Clamping

In situ patch clamp recordings are used for electrophysiological characterization of neurons in intact circuitry. In the Drosophila genetic model patch clamping is difficult because the CNS is small and surrounded by a robust sheath. This article describes the procedure to remove the sheath and clean neurons for subsequent patch clamp recordings.

Preparação de<em&gt; Drosophila</em&gt; Os neurônios centrais para<em&gt; In situ</em&gt; Patch aperto

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Gravação eletrofisiológicas na Drosophila Embrião

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Grampo patch Gravações de Embryonic Cells Zebrafish Mauthner

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at the Drosophila NMJ enables researchers to acquire synaptic information, encompassing both quantitative data on synapse abundance and detailed insights into their morphology. However, the diffuse distribution and limited visual range of the TEM present challenges for the ultrastructural analysis. This study introduces an innovative and efficient sample preparation method that surpasses the conventional approach. The procedure begins by placing a metal mesh at the base of a flat-bottomed bottle or test tube, followed by positioning fixed larvae samples onto the mesh. An additional mesh is placed over the samples, ensuring that they are positioned between the two meshes. The fixed samples are thoroughly dehydrated and infiltrated before proceeding with the embedding procedure. Then embedding of the samples in epoxy resin is performed in a flat sheet manner, which allows for the preparation of muscles for positioning and sectioning. Benefiting from these steps, all the muscles of Drosophila larvae can be visualized under light microscopy, therey facilitating subsequent positioning and sectioning. Excess resin is removed after locating the 6th and 7th muscles of body segments A2 and A3. Serial ultra-thin sectioning of the 6th or 7th muscle is performed.

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

This report provides a new sample preparation procedure for visualizing neuromuscular junctions in Drosophila Larvae. This method is more effective in preventing the curling of the samples compared to the traditional method and is particularly useful for Drosophila neuromuscular junction ultrastructural analysis.

Otimizando o processo de preparação de amostras para microscopia eletrônica de transmissão de junções neuromusculares em larvas de Drosophila

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at ...

Drosophila Neuromuscular Junction (NMJ) Quantification: A Method to Assess Synaptic Morphology and Function

Source: Castells-Nobau, A., et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/55395/" target="_blank">Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology</a>. J. Vis. Exp. (2017).
The Drosophila neuromuscular junction (NMJ) is used as a model to study the morphology and function of synapses. This video describes important features that researchers quantify to characterize the NMJ. The example protocol demonstrates a software able to perform the quantification automatically.

drosófila Quantificação da Junção Neuromuscular (NMJ): Um Método para Avaliar a Morfologia e Função Sináptica

Source: Castells-Nobau, A., et al. Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology. ...

Macropatch focal gravações das correntes sinápticas da junção Neuromuscular Larval da drosófila

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