Antes de comenzar preparar: <ul><li> Vagando yhird instar las larvas de Drosophila </li><li> HL3.1 (Modificado hemolinfa-como) una solución </li><li> Sylgard (caucho de silicona transparente) platos preparados en la disección pequeño (35 x 10 mm) placas petri de plástico con los métodos descritos por Brent y McCabe (2008) 3. </li><li> Cortar pernos disección corta </li><li> Estimular pipetas electrodo </li><li> Pipetas afiladas grabación </li></ul> HL3 Solución: <ol><li> Durante las disecciones y experimentos electrofisiológicos, larva se encuentran inmersos en HL3.1 solución 2 que contiene (en mM): NaCl 70, 5 KCl, 4 MgCl 2, 10 NaHCO 3, 5 trehalosa, sacarosa 115, y HEPES 5, pH 7,2. </li><li> Las concentraciones de CaCl 2 se añade a la solución HL3.1 para proporcionar la concentración requerida de Ca 2 + extracelular. <ul><li> Disecciones de las larvas se llevan a cabo a bajas concentraciones de Ca 2 + (para evitar la contracción muscular), utilizando HL3.1 + 0,25 mM CaCl 2, se mantienen en hielo. </li><li> Los experimentos electrofisiológicos suelen ser registrados en HL3.1 + 1 mM CaCl 2, pero esto puede ser ajustado 0,4 a 1 mM Ca 2 + según sea necesario. </li></ul></li><li> La solución es HL3.1 filtro esterilizado antes de su uso, y almacenadas a 4 ˚ C. </li></ol> Preparación de estimulación y registro de pipetas: <ol><li> Pipetas de grabación y estimulante se retiró con una Sutter P-2000 extractor de láser basado en micropipeta con los siguientes parámetros: calor = 390, filamento = 4, velocidad = 35, Delay = 200 y Pull = 0. </li><li> Electrodos de estimulación se preparan a partir de pared delgada capilares de vidrio de borosilicato con extremos anchos que eran un poco más grande que el ancho de los axones del motor roto. Los extremos son en forma de firepolished para darle un acabado suave (con una pipeta Narashiga pulidora), para minimizar el daño al nervio. Electrodos de estimulación se llena con una solución de baño (HL3.1 + 1 mM Ca 2 +). </li><li> Los electrodos de registro se preparan a partir de 1,2 mm de vidrio de borosilicato, que se retiró para formar una pipeta fuerte (30-60 mW), y lleno de KCl 3M. </li></ol> Parte 1: La disección de las larvas de Drosophila <ol><li> Larvas de tercer estadio errantes son disecados para exponer los músculos de la pared del cuerpo como se describió anteriormente 4. </li><li> Disecciones se realizan en HL3.1 + 0,25 mM Ca 2 + en las placas de silicona pequeño (35 x 10 mm). HL3.1 solución debe ser enfriado con hielo para la disección con el fin de anestesiar a la larva, y se mantiene en hielo antes y durante la disección. </li><li> Las larvas son disecados mediante los métodos descritos por Brent y McCabe (2008), que se ha modificado para los experimentos de electrofisiología y por la adición de 0,25 mM Ca 2 + a la solución de la disección HL3.1, y el uso de clavos cortos disección (~ 2 mm de longitud) que son menos probabilidades de golpear el objetivo del microscopio y de los electrodos durante un experimento. </li><li> Después de la disección, los axones motores de las larvas se cortan. Esto se hace con suavidad la celebración de la CNS y elevarlo un poco para los nervios periféricos que inervan los músculos posteriores al ganglio ventral se puede cortar sin dañar los músculos (Figura 1). El cerebro se retira. </li><li> La preparación de las larvas disecadas se lava dos veces con HL3.1 + 1 mM Ca 2 +. </li></ol><img src="/files/ftp_upload/1109/fig1.png" alt="Figura 1" /> Figura 1. Esquema que muestra los pasos a seguir en el corte de los axones de motor de las larvas de disección. El sistema nervioso central se eleva con las pinzas y los axones motores se cortan en la base del cerebro. Parte 2: registros intracelulares de las células musculares de larvas. <ol><li> Colocar la placa en el microscopio de disección de la electrofisiología y sumergir la preparación con HL3.1 + 1.5 mM Ca 2 + y fijar el electrodo de baño por lo que está en contacto con la solución. </li><li> Todos los experimentos se llevan a cabo en el músculo 6 en el tercer segmento abdominal (A3). Los nervios periféricos que inervan los músculos se estimulan con un electrodo de succión 5. </li><li> La posición del electrodo de estimulación en el primer plato para evitar las vibraciones cuando el electrodo intracelular está en su lugar. Coloque el electrodo de estimulación en las cercanías de la neurona motora inerva los músculos 6 y aplica la succión suave hasta que el nervio cortado está dentro de la pipeta de vidrio. Tenga cuidado de no estirar los nervios cuando se aplica succión. Aumentar la pipeta ligeramente por lo que no está en contacto con el músculo y la posición de modo que no es tirar de las fibras nerviosas cortadas (Figura 2). <img src="/files/ftp_upload/1109/fig2.png" alt="Figura 2" width="600" height="296" /> Figura 2. Un esquema de la musculatura y que inervan las neuronas motoras de un segmento abdominal de la larva de Drosophila. La posición de la pipeta y estimular los nervios cortados y el electrodo de registro en la posición de los músculos 6 se ilustran. </li><li> Intragrabaciones de celulares se hacen con microelectrodos fuerte lleno de 3 M KCl. La posición del electrodo de registro sobre el centro del músculo 6 en el tercer segmento abdominal (A3) y ajustar el offset de entrada para que se lea cero para la solución del baño. Baje lentamente el electrodo y el enfoque de los músculos bajo magnificación óptica de alta hasta que toque la superficie del músculo. Vea el osciloscopio para confirmar que el músculo ha sido penetrado. El potencial de reposo de la membrana debe ser de al menos 60 mV, o el animal no debe ser utilizado. Potenciales de placa terminal en miniatura esporádicos ahora debería ser visible. Salir de la celda se estabilice por un minuto antes de comenzar a grabar. </li><li> Los cambios en el potencial de membrana se detectan con una etapa de la cabeza Axón HS-2A y 2B Axoclamp un amplificador y grabado con Clampex v 8.2.0.235 (Axon Instruments). El 2B Axoclamp está interconectado a un equipo que ejecuta software pCLAMP y trabaja en conjunto con la interfaz de Digidata 1322A. </li><li> Las células se registraron durante 3 minutos sin ningún tipo de estímulos para medir las respuestas mEJP. Las huellas fueron analizados mediante el software de análisis de Mini (Synaptosoft, v 6.0.3) y la amplitud mEJP y frecuencia determinada. </li></ol> La estimulación del nervio: <ol><li> El extremo cortado de la neurona motora se estimula con una serie de pulsos de tensión cuadrados (0,3 ms de duración) a una intensidad que es suficiente tanto para los axones motores, para evocar una respuesta coherente en todo el músculo. </li><li> El estímulo se genera con el Maestro-8 generador de impulsos, que está programado para entregar pulsos continuamente de acuerdo a la duración y los tiempos de intervalo. </li><li> Espere cinco segundos entre los estímulos para la recuperación sináptica en la UNM. </li><li> Al determinar la fuerza del estímulo, comience con el nivel más bajo y aumentar la intensidad lentamente a lo largo de varios ensayos. La intensidad del estímulo mínimo que se traduce en una respuesta evocada deben ser utilizados en experimentos. </li><li> Cuando la intensidad del estímulo adecuado se alcanza, debe haber una EJP compuesto y una contracción muscular provocada por el estímulo. </li><li> Registro de por lo menos 10 potenciales evocados de cada músculo y el promedio de las amplitudes. </li></ol> Parte 3: Resultados de Representante <img src="/files/ftp_upload/1109/fig3.png" alt="la figura 3" width="600" height="358" /> Figura 3. Grabación Representante intracelular del músculo 6, que muestra el evocado EJP es la respuesta a la estimulación eléctrica del nervio segmentario, y los potenciales de placa terminal en miniatura esporádica, o de mEJP. EJP amplitudes en el músculo sano 6 de larvas de tipo salvaje, como Cantón S, son por lo general alrededor de 40 mV y las amplitudes mEJP entre 3.1 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>

Los métodos descritos aquí proporcionan una manera relativamente rápida y amplia para detectar los cambios en la función sináptica en la UNM. La capacidad de realizar los registros electrofisiológicos con animales intactos in vivo, y llevar a cabo manipulaciones genéticas o farmacológicas, que Drosophila un modelo animal ideal para investigar los aspectos fisiológicos y genéticos de la neurotransmisión. Dado que las células musculares son muy grandes, otros prefieren agregar un p...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<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

En este video, se describen los métodos electrofisiológicos para la grabación de la transmisión sináptica en la unión neuromuscular (UNM) de larvas de Drosophila. La larva sistema neuromuscular es una sinapsis modelo para el estudio de la fisiología sináptica y la neurotransmisión, y es una valiosa herramienta de investigación que ha definido la genética y es accesible a la manipulación experimental. Las larvas pueden ser diseccionados para exponer la pared del cuerpo musculatura, el sistema nervioso central, y los nervios periféricos. Los músculos de Drosophila y su patrón de inervación están bien caracterizados y los músculos son de fácil acceso para el registro intracelular. Los músculos individuales pueden ser identificados por su ubicación y orientación dentro de los 8 segmentos abdominales, cada una con 30 músculos dispuestos en un patrón que se repite en los segmentos A2 - A7. Disecados larvas de Drosophila son músculos delgados y el individuo y los haces de axones de neuronas motoras pueden ser visualizados por transiluminación<sup&gt; 1</sup&gt;. Construcciones transgénicas puede ser utilizada para etiquetar las células diana para la identificación visual o para la manipulación de los productos de genes en tejidos específicos. En las larvas, los potenciales excitatorios cruce (EJPs) se generan en respuesta a la liberación vesicular de glutamato de las neuronas motoras en la sinapsis. En las larvas de disección, el EJP se pueden registrar en el músculo con un electrodo intracelular. Los potenciales de acción puede ser artificialmente evocada en las neuronas motoras que se han cortado por detrás del ganglio ventral, elaborado en una pipeta de vidrio por succión suave y estimulado con un electrodo. Estas neuronas motoras tienen distintos umbrales de disparo cuando se estimula, y cuando el fuego al mismo tiempo, generan una respuesta en el músculo. Las señales transmitidas a través de la sinapsis MNJ se puede grabar en los músculos que la neuronas motoras inervan. El EJPs miniatura y potenciales de unión de excitación (mEJPs) son vistos como los cambios en el potencial de membrana. Respuestas electrofisiológicas se registran en la temperatura ambiente en la modificación mínima hemolinfa, como solución<sup&gt; 2</sup&gt; (HL3) que contiene 5 mg mM<sup&gt; 2 +</sup&gt; Y de 1,5 mM Ca<sup&gt; 2 +</sup&gt;. Los cambios en la amplitud de EJPs evocado puede indicar diferencias en la función sináptica y la estructura. Grabaciones digitalizadas son analizadas por EJP amplitud, frecuencia y amplitud mEJP, y el contenido cuántico.

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

A continuación se describen los métodos electrofisiológicos para la medición de la transmisión sináptica en la unión neuromuscular de larvas de Drosophila. Liberación evocada se inicia artificialmente mediante la estimulación de los axones de la neurona motora, y la transmisión a través de la UNM se puede medir por la respuesta postsináptica evocado en el músculo.

Los métodos electrofisiológicos para la grabación de los potenciales sinápticos de la UNM 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 vistas.  Columbia University College of Physicians and Surgeons. A continuación se describen los métodos electrofisiológicos para la medición de la transmisión sináptica en la unión neuromuscular de larvas de Drosophila. Liberación evocada se inicia artificialmente mediante la estimulación de los axones de la neurona motora, y la transmisión a través de la UNM se puede medir por la respuesta postsináptica evocado en el músculo.

Video: 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.

La disección de los discos imaginales de 3er estadio las larvas de Drosophila

Investigación

Biología

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 grabaciones de parche Capacidad de la pinza del cáliz

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 resolución de medición del comportamiento de olor-Driven en 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 NMJ Disección

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 NMJ inmunohistoquímica

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 estimulación mediada de los potenciales sinápticos en Drosophila Las uniones 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.

La recolección y preparación de Drosophila Los embriones de registro electrofisiológico y otros procedimientos

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.

Registro electrofisiológico en el Drosophila Embrión

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 y demostración de Fisiología en la UNM de los músculos inaugural cangrejo de río

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.

Imágenes in vivo de larvas de Drosophila intactos a nivel sub-celular Resolución

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