Financiado por el NIH (NS-18205).

1. Preparación de los peces para grabaciones de pares <ol><li> Antes de comenzar el procedimiento, preparar cinco soluciones clave, que incluyen: solución de baño, baño de solución con tricaína (MS222), solución de baño con formamida, la solución de las neuronas internas y la solución de los músculos internos. </li><li> A continuación, se recoge una sola larva del pez cebra entre las edades de 72 a 96 horas y el lugar en la solución de baño con tricaína. Dentro de un minuto de exposición a la anestesia, los peces no responden al tacto. </li><li> Coloque el pescado en la parte superior de un plato de plástico y, utilizando un microscopio estereoscópico, decapitar con una hoja de doble filo de la navaja. </li><li> La transferencia de los peces a una cámara de grabación de vidrio recubierto con Sylgard y lleno de solución del baño. Fijar en cada extremo mediante la inserción de electrólisis afilada pines de tungsteno a través de la notocorda. </li><li> Crear un colgajo de piel cerca del extremo rostral del pez con un alfiler que proporcionan un agarre adecuado para un par de pinzas finas. Retire la piel que lo recubre por agarrarlo cerca del pin rostral con un par de pinzas finas. Pele lentamente en dirección caudal para eliminar la piel. </li><li> Tratar a los peces con 3 ml de solución del baño con formamida durante 3-5 minutos para bloquear las contracciones musculares que pueden interferir con las grabaciones. Lavar 5 veces con solución de baño normal. Eliminar parte de la capa superficial de los músculos durante 5 a 6 segmentos con cuidado el desguace con el lado de un alfiler de tungsteno. </li><li> La transferencia de los peces a un microscopio vertical situado en una jaula de Faraday para proteger el ruido eléctrico. El microscopio está equipado con un escenario fijo, larga distancia de trabajo objetivo de 40x y zoom de 0,5 a 4 veces. El microscopio está montado en una etapa de traducción XY motorizado con el fin de mover la preparación entre el nervio y el músculo con alta potencia. El uso de la óptica de contraste de interferencia diferencial ayuda a visualizar las neuronas. </li><li> Por debajo de 0,5 x zoom, use un amplio diámetro de 15 micras pipeta para retirar el músculo que recubre y exponer la médula espinal. La presión negativa es aplicada a través de una jeringa de 3 ml desechables y las células musculares son eliminados uno por uno. Este procedimiento se repite de 5 a 6 segmentos dorsal del músculo esquelético. La misma variedad pipeta diámetro se utiliza para eliminar también las dos capas superiores de los músculos ventrales para obtener objetivos del músculo esquelético rápido. </li><li> Esto completa la preparación para las grabaciones de pares y el zoom microscopio se aumenta a aproximadamente 1,6 veces. </li></ol> 2. Grabaciones pares de CaP neurona motora y las células del músculo blanco <ol><li> Los segmentos de limpieza son escaneados para localizar las neuronas CaP. Cada hemi-segmento de la médula espinal contiene un gran diámetro, de 10 micras, Cap neurona motora. Este soma en forma de lágrima se encuentra superficialmente bajo la duramadre espinal, cerca del extremo más caudal de la frontera segmentaria. Una neurona de la PAC es elegido para la grabación y el músculo ventral en el segmento adyacente caudal se analiza para comprobar la integridad. </li><li> Dos electrodos se colocan parches para la grabación, uno para la neurona y una segunda para el músculo. El electrodo superior tiene una inclinación profunda de la penetración de la dura dura espinal y apertura de la punta de aproximadamente 2 micras. El electrodo inferior tiene una inclinación más pronunciada para la grabación de la abrazadera de baja resistencia a la tensión de los músculos esqueléticos </li><li> La posición del electrodo de la neurona en un ángulo de aproximadamente 30 grados para penetrar en la duramadre espinal, cerca de la mitad del segmento rostral a la neurona CaP seleccionado. Avanzar en el electrodo con un manipulador de accionamiento axial mientras se aplica una suave presión positiva continua de aproximadamente 60 milímetros de mercurio. A medida que el electrodo se rompe a través de la duramadre, la PAC, las neuronas pueden ser desplazados por la perfusión de los electrodos, lo que requiere un reajuste de la atención. </li><li> Cuando el electrodo toca la tapa de presión soma neuronal positiva se libera y por lo general resulta en la formación inmediata de un sello gigaohm. Sin embargo, en algunos casos en los que el sello no se forma, se hace necesario aplicar una suave presión negativa. Después de la formación del sello, el potencial del electrodo se ajusta a 80 mV y la aplicación de presión negativa se rompe la membrana celular. La neurona CaP se caracteriza por una resistencia de entrada de 140 a 180 Mega-ohmios y un potencial de acción que rebasa 40 milivoltios. Esta es la prueba mediante la inyección aumentando gradualmente los pasos de despolarización en pinza de corriente hasta que el potencial de acción se suscitó. </li><li> A continuación, el microscopio se traslada a los músculos ventrales con el traductor XY motorizado con alta potencia. Una célula de músculo rápido se elige en el campo de destino. </li><li> El electrodo del músculo se avanza hacia la célula del músculo blanco bajo presión positiva, que al ser liberado, forma un sello gigaohm. El potencial del electrodo se ajusta a -50 milivoltios para inactivar los canales de sodio y bajo succión suave de la membrana celular se rompió. Una caída en la resistencia de 100 a 200 Mega-ohmios y la repentina aparición de los grandes transitorios capacitivos entrada señales. </li><li> Para poner a pruebapara el registro de parejas, el motor de la neurona se despolariza por medio de inyecciones progresivamente mayor de la corriente hasta un potencial de acción se suscitó. En este punto, una corriente de placa terminal debe ser obtenido en el músculo. Fomento permanente de la motorneuron a 1 Hertz resultados en las corrientes de placa terminal sin fallos. A medida que la frecuencia del estímulo se incrementa a 100 Hertz las corrientes de placa terminal fluctúan en amplitud y se someten a reducción funcional dentro de los 10 segundos de estimulación. </li></ol> 3. Resultados representante Normalmente, la grabación de las neuronas es estable durante un máximo de una hora, pero las células musculares se deterioran como se refleja en un aumento de la resistencia en serie. Cuando esto ocurre, el experimento está bien terminado o de otra célula muscular es el parche una vez montado. Todas las grabaciones debe ser completado en una hora. <img src="/files/ftp_upload/2351/2351fig1.jpg" alt="Figura 1" /> Figura 1. Grabaciones del potencial de acción motorneuron (AP, arriba) y la placa terminal muscular asociada actual (EPC, abajo). El potencial de acción de una neurona de la PAC debería sobrepasar 40 mV y el EPC debería aumentar a 300 microsegundos y la decadencia a lo largo de un curso de tiempo exponencial con una constante de tiempo menor de 1 mseg. <table border="1"><tbody><tr><td> Nombre </td><td> Receta </td></tr><tr><td> Solución de baño (en mM) </td><td> 134 NaCl, 2,9 KCl, 2,1 CaCl 2, 1,2 MgCl 2, 10 glucosa, 10 Na-HEPES, pH 7,8 </td></tr><tr><td> Baño de solución con tricaína </td><td> Baño de solución que contiene 0,02% tricaína </td></tr><tr><td> Baño de solución con formamida </td><td> Baño de solución que contiene 2 millones de formamida </td></tr><tr><td> Neurona interna de la solución (en mM) </td><td> 115 K-gluconato, 15 KCl, 2 MgCl 2, 10 K-HEPES, 5 K-EGTA, 4 Mg-ATP, pH 7,2 </td></tr><tr><td> Los músculos internos de la solución (en mM) </td><td> 120 KCl, 10 K-HEPES, 5 BAPTA, pH 7,4 </td></tr></tbody></table> Tabla 1. Solutions.

<ol>
	<li>Fetcho, J. R., O'Malley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+networks+in+behaving+animals.">Imaging neuronal networks in behaving animals.</a> Current Opinion in Neurobiology. 7, 832-838 (1997).</li><li>Fetcho, J. R., S, H. i. g. a. s. h. i. j. i. m. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+and+genetic+approaches+toward+understanding+neuronal+circuits+in+zebrafish.">Optical and genetic approaches toward understanding neuronal circuits in zebrafish.</a> Integr Comp Biol. 44, 57-70 (2004).</li><li>Iba&#241;ez-Tallon, I., Wen, H., Miwa, J., Xing, J., Aslantas-Tekinay, A., Ono, F., Brehm, P., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tethering+naturally+occurring+peptide+toxins+for+cell+autonomous+modulation+of+ion+channels+and+receptors+in+vivo.">Tethering naturally occurring peptide toxins for cell autonomous modulation of ion channels and receptors in vivo.</a> Neuron. 43, 305-311 (2004).</li><li>Li, W., Ono, F., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+measurements+of+presynaptic+release+in+mutant+zebrafish+lacking+postsynaptic+receptors.">Optical measurements of presynaptic release in mutant zebrafish lacking postsynaptic receptors.</a> J. Neuroscience. , 23-10467 (2003).</li><li>Ono, F., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholine+receptors+direct+rapsyn+clusters+to+the+neuromuscular+synapse+in+zebrafish.">Acetylcholine receptors direct rapsyn clusters to the neuromuscular synapse in zebrafish.</a> J. Neuroscience. 24, 475-5481 (2004).</li><li>Ono, F., Shcherbatko, A., Higashijima, S., Fetcho, J., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paralytic+zebrafish+lacking+ACh+receptors+fail+to+localize+rapsyn+clusters+to+the+synapse.">Paralytic zebrafish lacking ACh receptors fail to localize rapsyn clusters to the synapse.</a> J. Neuroscience. 21, 5439-5448 (2001).</li><li>Ono, F., Shcherbatko, A., Higashijima, S., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+motility+mutant+twitch+once+reveals+new+roles+for+rapsyn+in+synaptic+function.">The zebrafish motility mutant twitch once reveals new roles for rapsyn in synaptic function.</a> J. Neuroscience. 22, 6491-6498 (2002).</li><li>Wang, M., Wen, H., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+neuromuscular+synapses+in+the+zebrafish+choline-acetyltransferase+mutant+bajan.">Function of neuromuscular synapses in the zebrafish choline-acetyltransferase mutant bajan.</a> J Neurophysiol. , 100-104 (2008).</li><li>Wen, H., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paired+motor-neuron+muscle+recordings+in+zebrafish+test+the+receptor+blockade+model+for+shaping+synaptic+current.">Paired motor-neuron muscle recordings in zebrafish test the receptor blockade model for shaping synaptic current.</a> J. Neuroscience. 25, 8104-8111 (2005).</li></ol>

Hemos estado utilizando el pez cebra grabaciones pares (Wen y Brehm, 2005), principalmente para estudiar los procesos de liberación de la vesícula y el reciclaje durante la estimulación de alta frecuencia. Este proceso se interrumpe en los mutantes de la motilidad en el que la transmisión sináptica normal se ve comprometida debido a mutaciones puntuales en los principales componentes sináptica. Por ejemplo, las mutaciones que alteran los agregados postsináptica de los receptores (Ono et al., 2001, 2002, 2004) y la síntesis del transmisor de pre-sináptica (Wang et al., 2008) como resultado de las Naciones Unidas coordinado natación. Grabaciones de pares de proporcionar la información que puede identificar la fuente del defecto funcional, vinculando así el comportamiento, genética y fisiología. Ahora debería ser posible diseccionar los procesos subyacentes a la transmisión sináptica a través de la expresión transitoria de la PAC motor neuronas usando promotores juiciosa. De esta manera genes dominantes negativos o mutados pueden ser expresados ​​específicamente en las neuronas y se determinó tanto las consecuencias del comportamiento y electrofisiológicos. La ventaja adicional que ofrece la configuración de célula entera permite la diálisis de la soma de la PAC con los agentes que potencialmente pueden alterar reciclaje de vesículas, como las toxinas clostridiales y los buffers de calcio. Debido a la corta distancia entre el soma y el músculo, la difusión debe ocurrir dentro del marco de tiempo de las grabaciones. El potencial de esta preparación ha comenzado a ser explotado. La transparencia de los peces en este lugar la edad y superficial de las sinapsis también facilita el uso de herramientas ópticas (Fletcho y O&#39;Malley, 1997; Fletcho y Higashijima, 2004). Hemos tenido mucho éxito en la medición de endo y exocitosis en los peces que viven con tintes FM y los indicadores genéticos, como Synaptophluorin (Li et al., 2003). Además, las toxinas conjugado fluorescente se puede utilizar para etiquetar las proteínas sinápticas de imágenes de peces vivos (Ibáñez-Tallon et al., 2004). Dada la simplicidad de esta preparación es una gran promesa para el futuro estudio.

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<td>Pneumatic transducer tester</td>
<td>&nbsp;</td>
<td>Fluke Biomedical Instruments</td>
<td>DPM1B</td>
<td>&nbsp;</td>
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<td>0.002 x 3 inch tungsten rod</td>
<td>&nbsp;</td>
<td>A-M Systems Inc</td>
<td>715000</td>
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<td>Sylgard 184 Elastomer</td>
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<td>Dow Corning Corp.</td>
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<td>The thickness of application will affect the DIC optics</td>
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paired patch clamp recordings from motor-neuron and target skeletal muscle in zebrafish

Las larvas de pez cebra representan el sistema de vertebrados primer modelo que permite la grabación simultánea de patch clamp de una neurona motora espinal y los músculos objetivo. Esto es una consecuencia directa de la accesibilidad a ambos tipos de células y su capacidad para distinguir visualmente el único de la PAC segmental neuronal motor sobre la base de la morfología y localización. Este video muestra los métodos microscópicos para identificar un CaP neurona motora y las células diana muscular, así como las metodologías para la grabación de cada tipo de célula. Identificación de la PAC neurona motora tipo se confirma mediante la cumplimentación tinte sea o por las características biofísicas, como forma de onda del potencial de acción y resistencia de entrada de la célula. Neurona motora grabaciones habitualmente duran una hora que permita a largo plazo a partir de múltiples grabaciones de las diferentes células del músculo blanco. El control sobre el patrón de activación neuronal motor permite realizar mediciones de la frecuencia de la dependencia de la transmisión sináptica en la unión neuromuscular. Debido al gran tamaño cuántico y el bajo nivel de ruido proporciona sujeción total voltaje de la célula, todos los eventos unitarios se pueden resolver en el músculo. Esta característica permite el estudio de las propiedades básicas sináptica como propiedades de liberación, reciclaje de vesículas, así como la depresión sináptica y la facilitación. Las ventajas que ofrece esta preparación en vivo eclipse de anteriores modelos de sistemas neuromuscular en la que estudiaron las neuronas motoras son generalmente estimuladas por electrodos extracelulares y los músculos son demasiado grandes para la abrazadera de toda mancha de la célula. La preparación del pez cebra puede ser objeto de combinar el análisis electrofisiológico con una amplia gama de enfoques, incluyendo las líneas transgénicas, caída morfolino, la intervención farmacológica y de imágenes in vivo. Estos enfoques, junto con el creciente número de modelos de enfermedad neuromuscular proporcionada por líneas mutantes de pez cebra, abrir la puerta a una nueva comprensión de los derechos humanos trastornos neuromusculares.

Watch this Scientific Journal Video about Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish at JoVE.com

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

Las larvas de pez cebra representan el sistema de vertebrados primer modelo que permite la grabación simultánea de patch clamp de una neurona motora espinal y el objetivo del músculo esquelético. Este video muestra los métodos microscópicos para identificar un CaP segmentaria neurona motora y las células diana muscular, así como las metodologías para la grabación de cada tipo de célula.

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.  ...

paired-patch-clamp-recordings-from-motor-neuron-target-skeletal

Research

JoVE Journal

Neuroscience

17.0K vistas.  Oregon Health and Sciences University. Las larvas de pez cebra representan el sistema de vertebrados primer modelo que permite la grabación simultánea de patch clamp de una neurona motora espinal y el objetivo del músculo esquelético. Este video muestra los métodos microscópicos para identificar un CaP segmentaria neurona motora y las células diana muscular, así como las metodologías para la grabación de cada tipo de célula.

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

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Grabaciones de patch clamp de neuronas de ratón de retina en una rebanada preparación adaptada a la oscuridad

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Investigación

Neurociencias

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

Grabaciones de patch clamp en Inner células del oído pelo Aislado de pez cebra

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Mitochondria are involved in many important cellular functions including metabolism, survival1, development and, calcium signaling2. Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death3.
The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase4-5. In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol6. In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL7 interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity8 and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion leak conductance) of the SMVs.

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.

<sub&gt; 1</sub&gt; F<sub&gt; O</sub&gt; ATPasa preparación de vesículas y técnica para realizar Patch clamp grabaciones de membranas de la vesícula submitocondriales

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

El aislamiento de las neuronas sensoriales de<em&gt; Aplysia californica</em&gt; Se Patch clamp grabaciones de Corrientes glutamatérgicos

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

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 ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Plenario de células patch-clamp grabaciones de Morphologically- e identificados-neuroquímicamente interneuronas del hipocampo

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Pareadas Grabaciones de células enteras en organotípicos de hipocampo rebanadas

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

Electrofisiológico y Caracterización Morfológica de microcircuitos neuronales en el infarto agudo rodajas de cerebro Usando Emparejados patch-clamp grabaciones

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Las grabaciones de células enteras Patch-clamp en rodajas de cerebro

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

Subcelulares patch-clamp grabaciones desde el dominio somatodendrítica de la sustancia nigra dopamina Neuronas

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...