Este trabajo fue apoyado por el NIH subvención EY17606 (APS).

1. Electrodos de preparar <ol><li> Prepare los electrodos de registro y referencia a los dos por inmersión en NaCl 1 M y la conducción de 15V entre ellos utilizando una onda sinusoidal de 1 Hz ~ 20 s. </li><li> Hacer los electrodos de registro utilizando un extractor de micropipeta (Sutter Instrumentos P-97). Parche de pipetas de utilizar un vidrio de borosilicato con un diámetro exterior de 1,2 mm y un diámetro interior de 0,69 mm (Sutter Instruments, Cat. # B120-69-10). Medir la resistencia en serie a través de la punta del electrodo. Basándose en el tamaño de las células diana elegir un diámetro punta de la pipeta, porque los cuerpos celulares de aproximadamente 20 micras de diámetro en el uso de ~ 5 mW de los cuerpos celulares ~ 5 micras de diámetro en el uso de ~ 12 mW. </li><li> Preparar el terreno / electrodos de referencia. Con una jeringa de llenar un tubo de polietileno de sección pequeña (Intramedic, PE205) con 1 mM de NaCl, que incluye un 3,5% de agar (Sigma, Cat. # A7002) en NaCl 1M. Coloque el puente de agar sobre el electrodo de referencia Ag / AgCl. </li></ol> 2. La preparación de soluciones <ol><li> Solución de baño externo, &quot;los medios de comunicación que contiene bicarbonato (1 litro): 1 botella de Ames Ames medio (Sigma, Cat. # A1420), 1,9 g de NaHCO 3, 7 ml de penicilina-estreptomicina (Sigma, Cat. # P0781) para evitar que las bacterias el crecimiento. Ajustar la osmolaridad a ~ 280 mOsm. </li><li> Corte de la solución, &quot;los medios de comunicación que contiene el tampón HEPES pH (1 L): 1 botella de Ames Ames Medio, 0,887 g de NaCl, 2,38 g de HEPES, 7 ml de penicilina-estreptomicina. Ajustar el pH a 7.4 usando ~ 1M NaOH y / o HCl 1M. Ajustar la osmolaridad a ~ 280 mOsm. </li><li> -Aspartato de potasio (K-Asp) solución de la pipeta interna (en mm): 125 K-aspartato, 10 KCl, 10 HEPES, 5 NMG-HEDTA, 0,5 CaCl 2, 1 ATP-Mg, 0,2 GTP-Mg, pH se ajusta a ~ 7.3 con NMG-OH, y la osmolaridad se ajusta a ~ 280 mOsm. </li><li> Respaldo en agar para la estabilización de la temperatura de gelificación bajo agarosa que contiene la retina (5% en peso), 7,5 g de agar (Sigma, Cat. # A7002) fundido en 150 ml de dH 2 O. </li></ol> 3. Día del experimento <ol><li> Descongele ~ 1,5 ml de K-Asp solución interna y el fluoróforo de su elección del congelador. Diluir el fluoróforo a la concentración adecuada, que debe ser determinada empíricamente. </li><li> Vierta los medios de comunicación de Ames contiene NaHCO3 (~ 200 ml) en el recipiente de almacenamiento a prueba de luz y luego colocarlo en el baño de agua (30-32 ° C) y equilibrar con un 5% de CO 2 / 95% O 2 gas </li><li> Llene la botella con ~ 110 ml de los medios de comunicación de Ames contiene HEPES, lugar en el hielo y equilibrarlo con el 100% de O 2. </li><li> Coloque el resto de Ames los medios de comunicación que contiene NaHCO3 en un depósito de calefacción en la parte superior de la jaula de Faraday, y equilibrar con CO2 al 5% / 95% de gas O 2. Parafilm puede ser usado para atrapar el gas en el espacio por encima de la solución. </li><li> Prepare la baja temperatura de gelificación de agarosa (3%) mediante la adición de 0,75 g (Sigma, Cat. # A0701) a 25 ml de los medios de comunicación de Ames con HEPES y calentar la solución a ~ 115 ° C para fundir la agarosa. Revuelva hasta que la solución clara, sin burbujas, y guardar en un baño de agua (~ 42 ° C). </li><li> Llenar una pipeta de grabación con la solución de K-Asp interna de la sonda de punta. </li></ol> 4. Comience Experimento <ol><li> Practicar la eutanasia con el ratón y eliminar rápidamente los ojos con tijeras curvas. Gafas de infrarrojos debe ser utilizado para todos los procedimientos para prevenir la decoloración del pigmento visual. </li><li> Lugar de los ojos en un pedazo de papel de filtro para evitar que se mueva, y el uso de una fina lámina adhesiva de doble cara, haga un corte en la córnea mientras se mantiene el ojo con las pinzas. </li><li> Tan pronto el globo ocular es perforado, que la transferencia de los ojos en un plato lleno de los medios de comunicación de Ames. </li><li> Utilizando la ranura de la córnea como un punto de entrada, unas tijeras pequeñas se utilizan para cortar las porciones restantes de la córnea, después de lo cual el cristalino y humor vítreo se puede quitar. Tenga cuidado de que la retina permanece unido a la ojera, y almacenar el ocular en la oscuridad a 32 ° C en los medios de comunicación equilibrada con 5% de CO 2 / 95% O 2 de Ames. </li></ol> 5. Rebanar <ol><li> Hemisect uno de los oculares y disociar la retina del epitelio pigmentario de la retina. Quitar los bordes de la retina para que el tejido que se acueste. </li><li> Use una pequeña pipeta Pasteur de vidrio con una bombilla de látex para llenar una caja de Petri de 35 mm con agar fundido y arrastre a través de las pinzas a través del agar para comprobar su consistencia. </li><li> Cuando se puede empezar a ver el agar solidifique: <ol class="lalpha"><li> La transferencia de la retina en la parte superior del agar. </li><li> Use un trozo retorcido de Kimwipe para absorber el exceso de solución alrededor de la retina. </li><li> Colocar 2-3 gotas de Agar directamente sobre la retina y poner rápidamente un cilindro de plástico en la retina para formar un muro alrededor de él. Después dejar agar fundido adicionales a los lados del cilindro de plástico, mientras que el centrado de la retina en el agar. </li><li> La transferencia de la placa de Petri to un baño de agua helada para enfriar. </li></ol></li><li> Después de aproximadamente 45 segundos quitar el tejido del baño de agua helada y el uso de un émbolo para extruir el agar bloque del cilindro de plástico, presionando por el lado más alejado de la retina. </li><li> Use la hoja de afeitar de un solo lado para cortar un pequeño bloque de agar que contiene la retina y el pegamento del bloque en su lugar contra el tope de agar en la platina. </li><li> La transferencia de la platina hasta la bandeja vibrante microtomo y se vierte el hielo frío HEPES Ames en la bandeja que permite el bloque con la retina a estar completamente sumergido. </li><li> Rebanadas de la retina con un espesor de ~ 200 micras se pueden cortar a la tasa máxima de hoja de vibración y una velocidad de hoja anterior conjunto de tal forma que tarda de 4 a 5 segundos para cortar a través de la retina en cada rebanada. </li><li> Use una hoja de bisturí N º 11 para quitar cada una rebanada útil a la vez </li><li> Rebanadas de mercancía que contenga la retina se colocan en cámaras de grabación y son cargados con anclas de corte con hilo de nylon cruzarlas. Los hilos de nylon mantenga pulsado el agar que rodea la retina, dejando en la retina sin obstáculos para las grabaciones. </li><li> La transferencia de la cámara de grabación a la platina del microscopio en posición vertical, cerca de la cortina y encienda la fuente de luz infrarroja para ver el corte en una cámara sensible al infrarrojo. </li></ol> 6. Grabación <ol><li> Una vez que una buena tajada de retina (fotorreceptores intactos y con el ángulo de corte apropiado) se identifica iniciar la perfusión de la retina a un ritmo superior a 5 mL / min con los medios de comunicación climatizada de Ames que se calienta a 35 37 ° C y se equilibró con 5% de CO 2 / 95% O 2. </li><li> Algunos daños en la superficie puede ser evidente debido al proceso de corte. Esta capa delgada de células por lo general se puede eliminar con una pipeta con una punta rota, y con suavidad chupando los cuerpos de las células muertas. Esto normalmente se revelan las células vivas por debajo. Identificar un cuerpo saludable de las células, cuya posición se encuentra en los estratos de interés. </li><li> Llene una micropipeta con la solución interna KAsp y aplicar positiva (hacia fuera) la presión a través de la punta de la pipeta, y bajar la punta de la pipeta en la cámara de grabación y hasta el nivel de la célula mediante un micromanipulador. </li><li> Mueva la punta de la pipeta de tal manera que los hoyuelos de la membrana celular, y aplicar suavemente negativa (hacia adentro) la presión para hacer un sello de alta resistencia. De células enteras modo se puede lograr mediante la aplicación de presión negativa para el electrodo de entrar en la célula. </li><li> Estimular el tejido de la retina mediante LEDs puede evocar respuestas a la luz. </li><li> Fotografía de la célula de la cual se realizó la grabación, evocando la fluorescencia de los fluoróforos en la solución de la pipeta interna. </li></ol> 7. Resultados representante <img src="/files/ftp_upload/2107/2107fig1.jpg" alt="Figura 1" /> Figura 1. Aquí, la luz potenciales evocados de una adaptada a la oscuridad de neuronas retinianas de destellos de luz para aumentar la fuerza se muestran. <img src="/files/ftp_upload/2107/2107fig2.jpg" alt="Figura 2" /> Figura 2. Imagen de fluorescencia de una rebanada de la retina donde las células se han llenado con el fluoróforo, Lucifer amarillo. Fluorescencia evocado muestra la morfología de las células de las cuales se hicieron las grabaciones de parches.

<ol>
	<li>Weblin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+along+and+between+rods+in+the+tiger+salamander+retina.">Transmission along and between rods in the tiger salamander retina.</a> J Physiol. 280, 449-470 (1978).</li><li>Wu, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+connections+among+retina+neurons+in+living+slices.">Synaptic connections among retina neurons in living slices.</a> J Neurosci Methods. 20, 139-149 (1987).</li><li>Rieke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+contrast+adaptation+in+salamander+bipolar+cell.">Temporal contrast adaptation in salamander bipolar cell.</a> J Neurosci. 21, 9445-9454 (2001).</li><li>Armstrong-Gold, C., &amp;amp, R. i. e. k. e., F, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bandpass+filtering+at+the+rod+to+second-order+cell+synapse+in+salamander+(Ambystoma+tigrinum)+retina.">Bandpass filtering at the rod to second-order cell synapse in salamander (Ambystoma tigrinum) retina.</a> J Neurosci. 23, 3796-3806 (2003).</li><li>Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fundamental+plan+of+the+retina.">The fundamental plan of the retina.</a> Nat Neurosci. 4, 877-886 (2001).</li><li>Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+processing+in+the+mammalian+retina.">Parallel processing in the mammalian retina.</a> Nat Rev Neurosci. 5, 747-757 (2004).</li></ol>

Grabación de sector a partir de las retinas de vertebrados se han hecho durante décadas 1,2, y han tenido bastante éxito en la prestación de un mejor entendimiento de cómo los circuitos de la retina codifica la luz incidente. Las ventajas del corte con un micrótomo de vibración en lugar de hacerlo directamente con una hoja de afeitar es que las rodajas de la retina incurrir en un menor daño en la superficie. Con una pipeta de succión que es fácil de quitar estos daños y las células diana sano deba...

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<td>Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>P-97</td>
<td>&nbsp;</td>
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<td>Borosilicate glass</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>B120-69-10</td>
<td>&nbsp;</td>
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<td>Polyethylene tubing</td>
<td>&nbsp;</td>
<td>Intramedic</td>
<td>PE205</td>
<td>&nbsp;</td>
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<td>Agar</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7002</td>
<td>&nbsp;</td>
<tr>
<td>Low gelling temp agarose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A0701</td>
<td>&nbsp;</td>
<tr>
<td>Ames' Medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A1420</td>
<td>&nbsp;</td>
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<td>Penicillin-Streptomycin</td>
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patch clamp recordings from mouse retinal neurons in a dark-adapted slice preparation

Nuestra experiencia visual se inicia cuando el pigmento visual en nuestra fotorreceptores de la retina absorbe fotones de energía de la luz e inicia una cascada de eventos intracelulares que llevan a la clausura de nucleótidos cíclicos, los canales con compuertas en la membrana celular. El cambio resultante en el potencial de membrana a su vez conduce a una reducción en la cantidad de la liberación de neurotransmisores de los dos terminales de la barra y cono sináptica. Para medir cómo el cambio de luz-evocada en el potencial de membrana del fotorreceptor conduce a la actividad posterior de la retina, los científicos han hecho registros electrofisiológicos de las preparaciones parte de la retina para 1,2 años. En el pasado estos cortes se han reducido de forma manual con una hoja de afeitar en el tejido de la retina que se une a un papel de filtro, en los últimos años otro método de corte ha sido desarrollado por el cual el tejido retinal está incrustado en agar de baja temperatura de gelificación y en rodajas en una solución fría con un vibración micrótomo 3,4. Esta preparación produce cortes de la retina con menos daños en la superficie y las respuestas evocadas por la luz muy fuerte. Aquí se documenta cómo este procedimiento puede realizarse bajo la luz infrarroja para evitar la decoloración del pigmento visual.

Watch this Scientific Journal Video about Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation at JoVE.com

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

Aquí se describe un procedimiento para la generación adaptada a la oscuridad rebanadas de la retina del ratón de los registros electrofisiológicos.

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 ...

patch-clamp-recordings-from-mouse-retinal-neurons-dark-adapted-slice

Research

JoVE Journal

Neuroscience

14.3K vistas.  University of Southern California. Aquí se describe un procedimiento para la generación adaptada a la oscuridad rebanadas de la retina del ratón de los registros electrofisiológicos.

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

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

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 ...

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

Simultáneas registros de células de fotorreceptores y las neuronas de segundo orden en una rebanada preparación retina Anfibios

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

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 ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

Grabación Luz evocó-Respuestas postsinápticos en las neuronas en adaptadas oscuro, Preparaciones ratón retina Slice Uso de técnicas Patch Clamp

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

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 ...