El apoyo de la beca del NIH R01 MH 099557

1. fabricación de electrodos de grabación <ol><li>tira de los electrodos de vidrio <ol><li>uso el siguiente protocolo para el tirador de microelectrodo (véase Tabla de materiales): <ol><li>línea 1: calor Tire de 510 - velocidad 30 tiempo 250; Línea 2: Calor tire 490 - velocidad 30 tiempo 250. 
			Nota: Unidades de tiempo corresponden a 0,5 m por unidad; las otras unidades son relativas. El valor del calor debe ajustarse para cada filamento, después de realiza la prueba de rampa.</li>
		</ol></li> <li>Uso de un microscopio (35 aumentos) para que el diámetro interior del electrodo tirado está en la gama de 7-10 μm ( figura 1A). Almacenar los tubos capilares en recipientes herméticamente cerrados para evitar la acumulación de polvo.</li>
	</ol></li> <li>Fuego pulido <ol><li>tubos capilares fuego polaco (80-90% del valor máximo de calor para 1-2 s) usando una micro-forja (véase Tabla de materiales). El diámetro interior final del electrodo pulido esté 5 μm ( figura 1A) 27.</li>
	</ol></li> <li>Flexión Nota: se hacen dos curvas para colocar el electrodo en la parte superior del músculo bajo un objetivo de gran aumento.
	<ol><li>Uso el aparato mostrado en la figura 1B. Fijar el electrodo en el manipulador y coloque la punta sobre el filamento, no tocar lo</li>
		<li>Conjunto valor a 60-70% del máximo del calor y apriete el pedal de la fragua de micro de 1-2 s (véase Tabla de materiales) para calentar el filamento. Utilice una aguja en forma de L para suavemente tire hacia abajo la punta del electrodo ( figura 1B ampliada). Realizar la curva en aproximadamente 90 o ( figura 1).</li>
		<li>Sostener el electrodo sobre la llama de la antorcha a mano usando pinzas y hacer la segunda curva a una distancia de 7-10 mm de la primera curva y en un ángulo de aproximadamente 120 o ( figura 1).</li>
	</ol></li></ol> <img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56493/56493fig1.jpg" /> figura 1. Pasos finales de la fabricación de una micropipeta. (A) electrodo consejos después de tirar y fuego pulido. (B.1) la configuración para el doblez de la punta. (B.2) el área de caja se muestra ampliada a la derecha. El electrodo y el filamento se fijan para que la punta del electrodo está situado un poco por encima el cable y no tocar. (C.1) el electrodo de la grabación. (C.2) el área de caja se muestra ampliada a la derecha. La primera curva tiene un ángulo de aproximadamente 90° y la distancia entre la primera curva y la punta del electrodo es de aproximadamente 1 mm. escala de la barra = 3 mm. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig1large.jpg" target="_blank"> Haga clic aquí para ver una versión más grande de esta figura.</a>
 2. pasos preparatorios adicionales <ol><li>Prepare la solución de heamolymph-like (HL3) (en mM): NaCl 70, 5 KCl, 20 MgCl 2, 10 NaHCO 3, 5 trehalosa, sacarosa 115, 5 HEPES y 1 mM CaCl 2; ajustar el pH a 7.3 - 7.4. mantener la solución en la nevera y hacer fresco cada semana.</li>
	<li>Hacer pipetas de estimulación la misma manera como grabación de vidrio pipetas, excepto que no es necesario doblarlas. 
	Nota: La preparación de los electrodos de estímulo se describe detalladamente en 28 y 27. El diámetro final después de pulir de fuego debe estar en el rango de 5-7 μm.</li>
	<li>Insertar una pipeta de estimulación en un soporte de microelectrodo conectada a una jeringa de.</li>
	<li>Placas de disección fabricación de pequeños platos de petri (35 x 10 mm) recubiertos con epoxi de caucho de silicona (véase Tabla de materiales) como se describe en 28. 
	Nota: El caucho de silicón debe ser completamente endurecido antes de uso.</li>
	<li>Elegir un errante larvas de tercer estadio y diseccionar como se describe en 27 , 28 , 29 , 30. 
	Nota: Para visualizar los botones, usar la cepa mosca CD8-GFP (véase Tabla de materiales) <ol><li>usar fórceps (véase Tabla de materiales), recoger las larvas de estadio 3 rd de un frasco de.</li>
		<li>Clavija las larvas, colocando el primer pasador posterior y la otra clavija anterior (cierre a ganchos de la boca).</li>
		<li>HL3 Agregar solución.</li>
		<li>Hacer un corte con tijeras de primavera (véase Tabla de materiales) desde el pasador superior a la inferior en el lado dorsal de las larvas de.</li>
		<li>Pin el filete de larvas, colocando 2 clavijas adicionales en los lados izquierdo y derecho de las larvas de.</li>
		<li>Quitar las tripas y traquear usando fórceps.</li>
		<li>Cortar los nervios a las afueras de la cuerda ventral del nervio con unas tijeras de muelle.</li>
	</ol></li></ol> 3. Grabaciones eléctricas de EJCs <img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56493/56493fig2.jpg" /> figura 2. La configuración de grabación. La muestra cubrió para el silicio recubierto de Petri (flecha) se coloca sobre el escenario móvil del microscopio compuesto vertical equipado con epifluorescencia, un objetivo de gran aumento y dos micromanipuladores NMJ. Se coloca el microscopio en una mesa antivibración. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig2large.jpg" target="_blank"> haga clic aquí para ver una versión más grande de esta figura.</a>
<ol><li>Grabación <ol><li>lugar el plato petri con la preparación sobre la platina del microscopio ( figura 2). Colocar el electrodo de referencia en el baño.</li>
		<li>Rellenar el electrodo de la grabación con solución de HL3. Bajo el objetivo de 10 x (véase Tabla de materiales), sumergir el electrodo en el baño y coloque sobre los músculos 6 y 7 de los segmentos abdominales 2, 3 o 4 con el micromanipulador () (véase Tabla de materiales) figura 3 A.1 y A.2).</li>
		<li>Cambiar el objetivo a x 60 (véase Tabla de materiales). Se centran en el área de interés usando epi-fluorescencia u óptica CID. Coloque la punta del electrodo en la parte superior bouton sináptico ( figura 3 B.1-3).</li>
		<li>Presionar el electrodo muy suavemente en el músculo. La presión excesiva puede dañar el NMJ o inducir un aumento en la actividad sináptica espontánea. Asegúrese de que la punta del electrodo no es obstruido - si es, reemplazar it</li>
		<li>Encender el amplificador, Junta de A/D y el equipo.</li>
		<li>Elegir el modo de pinza de voltaje del amplificador de.</li>
		<li>Iniciar el software de adquisición y elegir la &#39; boquete-libre &#39; modo de.</li>
		<li>Observar la aparición de mEJCs en la pantalla del ordenador.</li>
		<li>Asegurarse de que la amplitud de la mEJCs está en el rango de 0.2 - 0.7 na 
		Nota: EJCs más pequeño indican que el electrodo de la grabación tiene defectos o que no esté posicionado correctamente.</li>
	</ol></li></ol> <img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56493/56493fig3.jpg" /> figura 3. Visualización de los botones sinápticos. (A) A brightfield imagen de un hemi-segmento bajo 10 aumentos (A.1) y el área ampliada en caja mostrando los músculos 6 y 7 (A.2, la flecha marca el electrodo de la grabación). Barra de escala = 50 μm. (B) botones sinápticos se visualizan en una línea de Drosophila con un marcador neuronal genéticamente codificado (CD8-GFP) usando proyección de imagen de epi-fluorescencia (B.1) o DIC óptica (B.2). Imágenes son tomadas con el 60 x objetivo y el cubo de filtro para la proyección de imagen de GFP (véase Tabla de materiales). Synaptic botones están marcados con flechas, y B.3 muestra una superposición de imágenes DIC y fluorescentes. Barra de escala = 10 μm. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig3large.jpg" target="_blank"> haga clic aquí para ver una versión más grande de esta figura.</a>
<ol start="2"> <li>estimulación <ol><li>utilizando un micromanipulador, bajo un control visual, coloque el electrodo de estimulación cerca el axón inervan segmentos abdominales 2-4.</li>
		<li>Aplicar presión negativa tirando el émbolo de la jeringa conectada al cable porta electrodo, por lo que el axón se tira dentro del electrodo ( figura 2).</li>
		<li>Activar el estimulador. Gire el mando de la unidad de aislamiento (véase Tabla de materiales) para establecer una corriente de cero y luego irá aumentando progresivamente, hasta que aparezcan EJCs (o hasta que se alcanza el umbral).</li>
		<li>Realizar la estimulación en un régimen del suprathreshold, con la estimulación actual aumenta aproximadamente dos veces, en comparación con el umbral para la observación de los EJCs. 
		Nota: En nuestra experiencia, tal intensidad de estímulo es óptimo para evitar fallos de potencial de acción y potencial de acción de la leña. Por ejemplo, si EJCs aparecen en la corriente de estimulación de 0,2 mA, utilice una corriente de 0,4 mA durante todo el experimento.</li>
	</ol></li> <li>Resistencia <ol><li>Mida la resistencia del sello del electrodo de macropatch grabación girando el interruptor de la resistencia de electrodo del amplificador a &#34; prueba del sello &#34; posición. Observar que se mostrará el valor de la resistencia del sello en GΩ en el &#34; actual &#34; ventana.</li>
		<li>Asegúrese de que la resistencia del sello está en el rango de 0.5 - 2 M &#8486;. Valores fuera de este rango indican que el electrodo es no bien pulido o no debidamente colocado.</li>
		<li>Controlar la resistencia del sello a lo largo de la grabación, asegurándose de que permanece constante durante todo el experimento.</li>
	</ol></li></ol> 4. Análisis <ol><li>analizar grabaciones empleando personalizado software para el análisis (véase Tabla de materiales). 
	Nota: Nuestro laboratorio utiliza software interno turísticos 31, que está modificado para requisitos particulares para la detección de los EJCs y mEJCs grabados focal ( figura 4). Este software incluye un filtro digital gaussiano y permite la detección de picos cuántico de superposición cuántico múltiples eventos ( Figura 4A). Otros enfoques se describen en 17.</li>
</ol> <img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56493/56493fig4.jpg" /> figura 4. Análisis cuántico. Detección de mEJCs (A) y (B) EJCs por software turísticos. La zona de eventos está marcada en verde y picos están marcados por las puntas de flecha rojos. <a href="//ecsource.jove.com/files/ftp_upload/56493/56493fig4large.jpg" target="_blank"> haga clic aquí para ver una versión más grande de esta 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>

Los autores no tienen nada que revelar.

Drosophila representa un organismo modelo ventajoso para el estudio de la transmisión sináptica. Varias configuraciones de grabación se han utilizado en el NMJ larvas, incluyendo grabaciones intracelulares de potenciales sinápticos, grabaciones de corrientes sinápticas y voltaje de dos electrodos abrazadera33,34, macropatch focal grabaciones de corrientes sinápticas que se describe aquí. La última técnica permite la cuantificación precisa de la...

Drosophila es un sistema modelo excelente para estudiar los mecanismos moleculares de control de la transmisión sináptica. El sistema neuromuscular de Drosophila es glutamatérgica, y por lo tanto, la Unión neuromuscular de Drosophila (NMJ) puede utilizarse para estudiar las características conservadas de liberación glutamatérgica. Desde Jan y de Jan estudio1, las larvas de tercer estadio se ha utilizado ampliamente para estudiar la transmisión sináptica espontánea y evocada mediante el control de los potenciales excitatorios cruce (EJPs) o corrientes (EJCs). EJPs comúnmente se registran intracelular con un micro-electrodo de vidrio afilado, y reflejan la actividad de la NMJ entera, incluyendo todos los botones haciendo sinapsis en la fibra muscular determinada.
Por el contrario, puede registrar la actividad de un número limitado de los sitios de liberación focal colocando una punta de micropipeta cerca terminales neuronales o varicosidades sinápticas. Esta técnica fue empleada originalmente por Katz y Miledi2y grabaciones extracelulares focal han sido empleadas con éxito en varias preparaciones de NMJ, incluyendo rana3,4,5, ratón6 , 7 , 8, crustáceos9,10,11,12,13,14,15,16y Drosophila17,18,19,20,21,22,23. Este enfoque fue desarrollado por Dudel, que había optimizado macropatch recodificación electrodos24,25. En la implementación de Dudel, esta técnica muy igualados con el método de la abrazadera del remiendo flojo26.
La NMJ de Drosophila larvas ha definido claramente los botones sinápticos, y líneas transgénicas con las etiquetas fluorescentes neuronales genéticamente codificados (véase Tabla de materiales) están disponibles. Estas ventajas nos permitieron grabar EJCs y mEJCs de un bouton sináptico seleccionado20,21,22. Aquí, describimos esta técnica en detalle.

<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

Unión neuromuscular de Drosophila (NMJ) es un sistema modelo excelente para estudiar la transmisión sináptica glutamatérgica. Describimos la técnica de las grabaciones de macropatch focal de corrientes sinápticas de botones visualizados en la NMJ de Drosophila larvas. Esta técnica requiere fabricación modificada para requisitos particulares de grabación micropipetas, así como un microscopio compuesto equipado con un gran aumento, objetivo de inmersión de agua de larga distancia, óptica de interferencia diferencial (DIC) de contraste y un fluorescente accesorio. El electrodo de registro se coloca en la parte superior de un seleccionado bouton sináptico visualizado con DIC óptica, epi-fluorescencia o ambos. La ventaja de esta técnica es que permite seguimiento de la actividad sináptica de un número limitado de sitios de liberación. El electrodo de la grabación tiene un diámetro de varias micras, y los sitios de liberación colocados fuera del borde del electrodo no afectar significativamente las corrientes registradas. Las corrientes sinápticas grabadas tienen cinética rápida y pueden ser fácilmente resuelto. Estas ventajas son especialmente importantes para los estudios de líneas de mosca mutantes con mayor actividad sináptica espontánea o asincrónica.

Grabaciones de macropatch focal permiten monitoreo actividad sináptica de las botones sinápticos (figura 5). Cuando el electrodo se coloca en la parte superior un bouton sináptico (figura 5A, sitio 1), el grabado mEJCs (figura 5, Página 1) tienen una amplitud significativamente superior al nivel de ruido y sostenido aumento de las fases (en un rango de los milisegundos). Cuando el electrodo de la...

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

Grabaciones de Macropatch focal de corrientes sinápticas de la Unión Neuromuscular larvaria de Drosophila

Las corrientes sinápticas pueden grabarse focal de visualizadas botones sinápticos en la Drosophila tercer instar larvas Unión neuromuscular. Esta técnica permite la monitorización de la actividad de una sola bouton sináptico.

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 vistas.  Wayne State University. Las corrientes sinápticas pueden grabarse focal de visualizadas botones sinápticos en la Drosophila tercer instar larvas Unión neuromuscular. Esta técnica permite la monitorización de la actividad de una sola bouton sináptico.

Video: 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.

Emparejados grabaciones patch clamp de neurona motora y el músculo esquelético de destino en el pez cebra

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

Investigación

Neurociencias

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.

La disección y la imagen de zonas activas en el Drosophila La unión neuromuscular

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

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.

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. ...

Biología

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 ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Disección y de grabación de la unión neuromuscular C. Elegans

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

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.

Preparación de<em&gt; Drosophila</em&gt; Las neuronas centrales para<em&gt; In situ</em&gt; Parche de sujeción

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.

Registro electrofisiológico en el Drosophila Embrión

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.

Patch Clamp Las grabaciones de las células embrionarias de pez cebra 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.

Optimización del proceso de preparación de muestras para la microscopía electrónica de transmisión de uniones neuromusculares en 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.

Drosophila Cuantificación de la unión neuromuscular (NMJ): un método para evaluar la morfología y la función sináptica

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