Antes de iniciar preparar: <ul><li> Wandering yhird instar as larvas Drosophila </li><li> HL3.1 solução (Modificado hemolinfa-like) </li><li> Placas Sylgard (borracha de silicone transparente) dissecção preparado em pequena (35 x 10 mm) pratos de plástico petri usando métodos descritos por Brent e McCabe (2008) 3. </li><li> Cortar pinos dissecção curta </li><li> Estimulando pipetas eletrodo </li><li> Afiada pipetas de gravação </li></ul> HL3 Solução: <ol><li> Durante as dissecções e experimentos eletrofisiológicos, larva são imersos em solução de 2 HL3.1 que contém (em mM): 70 NaCl, KCl 5, 4 MgCl 2, 10 NaHCO 3, 5 trealose, 115, sacarose e 5 HEPES, pH 7.2. </li><li> Concentrações de CaCl 2 são adicionados à solução HL3.1 para fornecer a concentração necessária de Ca 2 + extracelular. <ul><li> Dissecções larvas são realizados em baixa Ca 2 + concentrações (para evitar a contração muscular), utilizando HL3.1 + 0,25 mM CaCl 2, mantido em gelo. </li><li> Experimentos eletrofisiológicos são normalmente registrados em HL3.1 + 1 mM CaCl 2, mas isso pode ser ajustado 0,4-1 mM Ca 2 +, conforme necessário. </li></ul></li><li> A solução é HL3.1 filtro esterilizadas antes do uso, e armazenados a 4 ˚ C. </li></ol> Preparação de estimulação e registro pipetas: <ol><li> Pipetas de gravação e estimulantes são puxados com um P-2000 Sutter extrator Laser micropipeta com base utilizando as seguintes definições: Calor = 390, Filamento = 4 Velocity, = 35, Delay = 200 e Pull = 0. </li><li> Eletrodos estimulantes são preparados a partir de paredes finas de vidro de borosilicato capilares com extremidades largas que eram ligeiramente maior que a largura da axônios motores cortados. As extremidades moldadas são firepolished para dar um acabamento liso (usando uma pipeta Narashiga polidor), para minimizar os danos ao nervo. Eletrodos estimulantes são preenchidos com solução de banho (HL3.1 + 1 mM Ca 2 +). </li><li> Os eletrodos de gravação são preparados a partir de 1,2 milímetros de vidro de borosilicato, que é puxado para formar uma pipeta afiada (30-60 mohms), e preenchido com KCl 3M. </li></ol> Parte 1: Dissecção de larvas de Drosophila <ol><li> Larvas de terceiro estádio errantes são dissecados para expor os músculos da parede do corpo, conforme descrito anteriormente 4. </li><li> Dissecções são realizados em HL3.1 + 0,25 mM Ca 2 + em placas de silicone pequena (35 x 10 mm). HL3.1 solução deve ser gelada para dissecções, a fim de anestesiar a larva, e é mantido no gelo antes e durante a dissecção. </li><li> As larvas são dissecados usando métodos descritos por Brent e McCabe (2008), que é modificado para experimentos de eletrofisiologia, adicionando 0,25 mM Ca 2 + para a solução dissecção HL3.1, e utilizando pinos de dissecção de curta duração (~ 2 mm de comprimento) que são menos probabilidade de acertar a objetiva do microscópio e eletrodos durante um experimento. </li><li> Após dissecação, os axônios motores das larvas são cortados. Isto é feito com cuidado, segurando o SNC e elevá-la ligeiramente para os nervos periféricos que inervam os músculos posteriores ao gânglio ventral pode ser cortada sem danificar os músculos (Figura 1). O cérebro é então removido. </li><li> A preparação das larvas dissecadas é lavada duas vezes com HL3.1 + 1 mM Ca 2 +. </li></ol><img src="/files/ftp_upload/1109/fig1.png" alt="Figura 1" /> Figura 1. Esquemático mostrando as etapas envolvidas no corte os axônios motores de larvas dissecadas. O CNS é levantado com uma pinça e os axônios motores são cortadas na base do cérebro. Parte 2: gravações intracelular das células musculares larval. <ol><li> Coloque a placa de dissecção no microscópio eletrofisiologia e submergir a preparação com HL3.1 + 1,5 mM Ca 2 + e fixar o eletrodo banho assim que está em contato com a solução. </li><li> Todos os experimentos são realizados no músculo seis dentro do terceiro segmento abdominal (A3). Nervos periféricos que inervam os músculos são estimulados através de um eletrodo de sucção 5. </li><li> Posição do eletrodo de estimulação no primeiro prato, para evitar vibrações quando o eletrodo intracelular está no lugar. Coloque o eletrodo estimulando nas imediações do neurônio motor inerva músculo 6 e aplicar sucção suave até que o nervo corte está dentro da pipeta de vidro. Tenha cuidado para não esticar os nervos quando a sucção é aplicada. Elevar a pipeta ligeiramente por isso não está tocando o músculo e posicioná-lo por isso não é puxando as fibras nervosas cortadas (Figura 2). <img src="/files/ftp_upload/1109/fig2.png" alt="Figura 2" width="600" height="296" /> Figura 2. Um esquema da musculatura e dos neurônios motores, que inervam de um segmento abdominal da larva Drosophila. A posição da pipeta estimulante e nervos cortados eo eletrodo de gravação na posição em músculo 6 são ilustrados. </li><li> Intragravações de celulares são feitos com microeletrodos afiada cheia com 3 M KCl. Posição do eletrodo de registro acima do centro do músculo 6 no terceiro segmento abdominal (A3) e ajustar a entrada compensado assim que lê zero para a solução do banho. Abaixe lentamente o eletrodo e se aproximar do músculo sob ampliação ótica de alta até tocar a superfície do músculo. Assista o osciloscópio para confirmar que o músculo tenha sido penetrado. O potencial de repouso deve ser de pelo menos -60 mV, ou o animal não deve ser usado. Esporádicos potenciais de placa terminal em miniatura deverão ser agora visíveis. Deixe o celular para estabilizar por um minuto antes de começar a gravar. </li><li> Mudanças no potencial de membrana são detectados com um estágio de cabeça Axon HS-2A e 2B Axoclamp um amplificador e gravou com Clampex v 8.2.0.235 (Axon Instruments). A 2B Axoclamp é conectado a um computador que executa o software pCLAMP e funciona em conjunto com a interface Digidata 1322A. </li><li> As células foram gravadas por 3 minutos, sem qualquer estímulo para medir as respostas mEJP. Traços foram analisados ​​usando o software de análise Mini (Synaptosoft, v 6.0.3) e da amplitude mEJP e freqüência determinada. </li></ol> A estimulação do nervo: <ol><li> O final cortado do neurônio motor é estimulado com uma série de pulsos de voltagem quadrados (0,3 ms de duração) em uma intensidade que é suficiente para ambos os axônios motores, para evocar respostas consistentes em todo o músculo. </li><li> O estímulo é gerado com o Mestre-8 gerador de pulsos, que é programado para entregar pulsos continuamente de acordo com a duração e os tempos de intervalo. </li><li> Permitir que cinco segundos entre os estímulos para a recuperação sináptica no MNJ. </li><li> Ao determinar a força do estímulo, começar com o nível mais baixo e aumentar a intensidade lentamente ao longo de vários ensaios. A intensidade do estímulo mínimo que resulta em uma resposta evocada deve ser usado em experimentos. </li><li> Quando a intensidade do estímulo adequado é alcançado, deve haver uma EJP composto e uma contração muscular evocada pelo estímulo. </li><li> Registro de pelo menos 10 potenciais evocados de cada músculo e média as amplitudes. </li></ol> Parte 3: Resultados Representante <img src="/files/ftp_upload/1109/fig3.png" alt="figura 3" width="600" height="358" /> Figura 3. Intracelular gravação Representante do músculo 6 mostrando o evocado EJP é resposta à estimulação elétrica do nervo segmentar, e os potenciais de placa terminal em miniatura esporádicos, ou mEJP é. EJP amplitudes no músculo 6 de larvas do tipo selvagem saudáveis, como Canton S, são normalmente em torno de 40 mV e as amplitudes mEJP entre 1-3 mV.

<ol>
	<li>Atwood, H. L., Govind, C. K., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+ultrastructure+of+synaptic+terminals+on+ventral+longitudinal+abdominal+muscles+in+Drosophila+larvae.">Differential ultrastructure of synaptic terminals on ventral longitudinal abdominal muscles in Drosophila larvae.</a> J. Neurobiol. 24 (8), 1008-1024 (1993).</li><li>Feng, Y., Ueda, A., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+minimal+hemolymph-like+solution,+HL3.1,+for+physiological+recordings+at+the+neuromuscular+junctions+of+normal+and+mutant+Drosophila+larvae.">A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae.</a> J Neurogenet. 18 (2), 377-402 (2004).</li><li>Estes, P. S., Roos, J., van der Bliek, A., Kelly, R. B., Krishnan, K. S., Ramaswami, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traffic+of+dynamin+within+individual+Drosophila+synaptic+boutons+relative+to+compartment-specific+markers.">Traffic of dynamin within individual Drosophila synaptic boutons relative to compartment-specific markers.</a> J Neurosci. 16, 5443-5456 (1996).</li><li>Brent, J. R., Werner, K. M., 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=Protocol+for+dissection+of+Drosophila+larvae.">Protocol for dissection of Drosophila larvae.</a> J Vis Exp. , (2008).</li><li>Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+the+larval+neuromuscular+junction+in+Drosophila+melanogaster.">Properties of the larval neuromuscular junction in Drosophila melanogaster.</a> J Physiol (Lond). 262, 189-214 (1976).</li><li>Katz, L. C., Shatz, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+activity+and+the+construction+of+cortical+circuits.">Synaptic activity and the construction of cortical circuits.</a> Science. 274, 1133-1138 (1996).</li></ol>

Os métodos descritos aqui oferecem uma maneira relativamente rápida e ampla para detectar mudanças na função sináptica no MNJ. A capacidade de realizar gravações eletrofisiológicas usando animais intactos in vivo, e executar manipulações genéticas ou farmacológicas, fazer Drosophila um modelo animal ideal para investigar os aspectos fisiológicos e genéticos da neurotransmissão. Como as células musculares são muito grandes, alguns podem preferir adicionar um passo adicional ...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>Small Petri dishes (35 x 10 mm)</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>1008</td>
<td>&nbsp;</td>
<tr>
<td>SYLGARD 182 Silicone Elastomer Kit</td>
<td>&nbsp;</td>
<td>Dow Corning Corporation</td>
<td>3097366-1004</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting microscope</td>
<td>&nbsp;</td>
<td>Carl Zeiss</td>
<td>475002-9902</td>
<td>&nbsp;</td>
<tr>
<td>Light for microscope</td>
<td>&nbsp;</td>
<td>Schott</td>
<td>KLI500</td>
<td>&nbsp;</td>
<tr>
<td>Dissection pins</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-10</td>
<td>&nbsp;</td>
<tr>
<td>pClamp 9 software</td>
<td>&nbsp;</td>
<td>Axon CNS, Molecular Devices</td>
<td>PCLAMP 9 STANDARD</td>
<td>&nbsp;</td>
<tr>
<td>Dissection scissors: 3mm Vannas Spring Scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15000-0</td>
<td>&nbsp;</td>
<tr>
<td>Dumont SS Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11200-33</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11252-20</td>
<td>&nbsp;</td>
<tr>
<td>Thin-walled borosilicate glass capillaries, with filament (1.0 mm, 4 in)</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>TW100F-4</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass capillaries, with filament (1.2 mm, 4 in)</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>1B120F-4</td>
<td>&nbsp;</td>
<tr>
<td>Sutter P-2000 Laser Based Micropipette Puller </td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>Model P-2000</td>
<td>&nbsp;</td>
<tr>
<td>Pipette polisher</td>
<td>&nbsp;</td>
<td>Narishiga</td>
<td>MF-83</td>
<td>&nbsp;</td>
<tr>
<td>Axon HS-2A head stage</td>
<td>&nbsp;</td>
<td>Axon CNS, Molecular Devices</td>
<td>Model HS-2A</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulators</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>MP-85</td>
<td>&nbsp;</td>
<tr>
<td>Axoclamp 2B amplifier</td>
<td>&nbsp;</td>
<td>Axon CNS, Molecular Devices</td>
<td>AXOCLAMP 2B</td>
<td>&nbsp;</td>
<tr>
<td>Clampex Software</td>
<td>&nbsp;</td>
<td>Axon CNS, Molecular Devices</td>
<td>v 8.2.0.235</td>
<td>&nbsp;</td>
<tr>
<td>Mini analysis software. v 6.0.3</td>
<td>&nbsp;</td>
<td>Synaptosoft</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Brownlee Precision Amplifier</td>
<td>&nbsp;</td>
<td>Brownlee</td>
<td>Model 410</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Baker</td>
<td>4058-01</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>p-9333 </td>
<td>&nbsp;</td>
<tr>
<td>NaHCO3</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>s6297-1kg</td>
<td>&nbsp;</td>
<tr>
<td>Trelahose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>TO167</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>bp220-212 </td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>h-3375</td>
<td>&nbsp;</td>
<tr>
<td>MgCl-6H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>m2670-1kg </td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>c79-500</td>
<td>&nbsp;</td>
<tr>
<td>Master-8 Pulse Generator</td>
<td>&nbsp;</td>
<td>A.M.P.I</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vibration table for electrophysiology set up</td>
<td>&nbsp;</td>
<td>Technical manufacturing corporation</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Faraday Cage</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

electrophysiological methods for recording synaptic potentials from the nmj of drosophila larvae

Neste vídeo, descrevemos os métodos eletrofisiológicos para a gravação da transmissão sináptica na junção neuromuscular (MNJ) da larva Drosophila. O sistema neuromuscular é uma sinapse larval modelo para o estudo da fisiologia sináptica e neurotransmissão, e é um valioso instrumento de pesquisa que definiu a genética e é acessível à manipulação experimental. As larvas podem ser dissecado para expor o corpo musculatura da parede, sistema nervoso central, e os nervos periféricos. Os músculos de Drosophila e seu padrão de inervação estão bem caracterizadas e os músculos são de fácil acesso para gravação intracelular. Músculos individuais podem ser identificados por sua localização e orientação dentro do 8 segmentos abdominais, cada um com 30 músculos dispostos em um padrão que se repete em segmentos A2 - A7. Dissecados larvas de drosófila são músculos finos e individuais e feixes de axônios do neurônio motor pode ser visualizado por transiluminação<sup&gt; 1</sup&gt;. Construções de transgênicos pode ser usado para rotular as células-alvo para a identificação visual ou para a manipulação de produtos de genes em tecidos específicos. Em larvas, potenciais de junção excitatórios (EJPs) são gerados em resposta a liberação vesicular de glutamato do motoneurônios na sinapse. Em larvas dissecadas, o EJP pode ser gravado no músculo com um eletrodo intracelular. Potenciais de ação podem ser artificialmente evocado nos neurônios motores que foram cortados posterior ao gânglio ventral, atraído para uma pipeta de vidro por sucção suave e estimulado com um eletrodo. Estes neurônios motores têm distintas limiar de disparo quando estimulados, e quando eles atiram simultaneamente, geram uma resposta no músculo. Sinais transmitidos através da sinapse MNJ podem ser gravadas nos músculos que os neurônios motores inervam. O EJPs e miniatura potenciais de junção excitatórios (mEJPs) são vistas como mudanças no potencial de membrana. Respostas eletrofisiológicas são registrados a temperatura ambiente em solução de hemolinfa modificadas-como o mínimo<sup&gt; 2</sup&gt; (HL3) que contém 5 Mg mM<sup&gt; 2 +</sup&gt; E 1,5 mM Ca<sup&gt; 2 +</sup&gt;. Mudanças na amplitude de EJPs evocado pode indicar diferenças na função sináptica e estrutura. Gravações digitalizadas são analisadas para EJP amplitude, mEJP freqüência e amplitude e conteúdo quântica.

Watch this Scientific Journal Video about Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae at JoVE.com

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

Aqui nós descrevemos métodos eletrofisiológicos para medir a transmissão sináptica na junção neuromuscular de larva Drosophila. Liberação evocada é iniciado artificialmente estimulando os axônios do neurônio motor ea transmissão através do MNJ pode ser medida pela resposta pós-sináptica evocado no músculo.

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. ...

electrophysiological-methods-for-recording-synaptic-potentials-from

Research

JoVE Journal

Biology

20.8K visualizações.  Columbia University College of Physicians and Surgeons. Aqui nós descrevemos métodos eletrofisiológicos para medir a transmissão sináptica na junção neuromuscular de larva Drosophila. Liberação evocada é iniciado artificialmente estimulando os axônios do neurônio motor ea transmissão através do MNJ pode ser medida pela resposta pós-sináptica evocado no músculo.

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

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

Dissecação de discos imaginais de 3 larvas Drosophila Instar

Biologia

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

Métodos para Recordings patch-clamp Capacitância da Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

De alta resolução de Medição do Comportamento Odor-Driven em larvas de Drosophila

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

Drosophila larval Dissection MNJ

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

Drosophila larval MNJ Imunohistoquímica

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 ...

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

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. 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.

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

Colheita e Preparação Drosophila Os embriões para Gravação eletrofisiológicos e outros procedimentos

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

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

Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is still much to learn from this model preparation. In understanding the history one can appreciate why even today this NMJ still offers a rich playground to address questions regarding pre- and post-synaptic function and plasticity. The viability and ease of access to the terminal for intracellular as well as extracellular electrophysiology and imaging are significant advantages. The mechanisms behind the modulation of vesicular kinetics and fusion within the high- and low-output terminals are begging for investigation. The preparation also offers a testable model system for computational assessments and manipulations to examine key variables in theoretical models of synaptic function, for example calcium dynamics during short-term facilitation. The synaptic complexity of active zone and statistical nature of quantal release is also an open area for future investigation both experimentally and computationally.

The opener muscle of the crayfish leg is presented for its historical importance and experimental versatility in muscle phenotype, synaptic physiology and plasticity.

Ver histórico e demonstração Fisiologia da MNJ da Muscle Opener Crayfish

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

Na imagem in vivo de larvas Drosophila Intact no Sub-celular Resolução

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...