We thank Joung Jang and Xin Guan for technical assistance. We thank Dr. Rory McQuiston of Virginia Commonwealth University for setting up our first patch clamp rig and advices on experimental procedures. We thank Dr. Samuel Wu for suggestions on voltage clamp recording. The work is supported by NIH grants EY013811, EY022228 and a vision core grant EY002520. C-KC is the Alice R. McPherson Retina Research Foundation Endowed Chair at the Baylor College of Medicine.

Ética aprobación - los procedimientos que implican sujetos animales se realizaron de conformidad con las reglas y regulaciones de los Institutos Nacionales de Salud de los animales de investigación, tal como fue aprobado por el Cuidado de Animales institucional y el uso comité de Baylor College of Medicine. 1. externa y de soluciones internas <ol><li> Use la solución de Ringer de mamíferos durante la disección retina y como la solución externa de registro electrofisiológico posterior. Preparar la solución de Ringer mamífero de la solución madre 10x (sin calcio) en el día de la grabación, y añadir CaCl2 gota a gota, después de 15 minutos de carbogenation (95% de O2 y 5% de CO 2). La solución final 1x contiene (en mM): 120 NaCl, 5 KCl, 25 NaHCO 3, 0,8 Na 2 HPO 4, 0,1 NaH 2 PO 4, 2 CaCl 2, 1 MgSO 4 y 10 D-glucosa. </li><li> Use dos soluciones internas para caracterizar las oscilaciones SAC. Pruebala utilidad de las soluciones preparadas en las grabaciones de patch clamp de células enteras prolongados en CGR de ratón adulto y almacenar los lotes verificados en 500 ml de alícuotas a -20 ° C hasta su uso. <ol><li> Para la grabación de potencial de membrana de oscilación, utilice una solución interna a base de potasio que contiene (en mM): 125 K-gluconato, 8 NaCl, 4 ATP-Mg, 0,5 Li-GTP, 5 EGTA, 10 HEPES y 0,2% biocitina (w / v). Ajustar el pH a 7,3 con KOH. </li><li> Para la grabación de excitación y de las corrientes postsinápticas inhibitorias, utilice una solución interna a base de cesio que contiene (en mM): 100 Cs-metanosulfonato, 8 NaCl, 4 ATP-Mg, 0,5 Li-GTP, 5 EGTA, 10 HEPES y 0,2% biocitina ( Virginia Occidental). Ajustar el pH a 7,3 con CsOH. </li></ol></li></ol> 2. Preparación para el Día de la grabación <ol><li> Para preparar una membrana de nitrocelulosa con los agujeros abiertos, perforar manualmente la membrana con un golpeador personalizada a partir de una 16 G aguja de la jeringa romo y aplanar la membrana entre dos placas de vidrio limpias. Por su conjunto-montar retina, hacer un agujero central de 0,2 mm de diámetro y rodearlo de 4 agujeros más grandes que son 1-1,2 mm de diámetro. </li><li> Para hacer un filamento de material de relleno a medida para cargar solución interna en los electrodos, fundir una punta de pipeta de 10 l en el medio y alargar suavemente la parte fundida hasta que se enfría y se endurece. Justo antes de usar, usar una hoja de afeitar limpia para cortar el filamento hasta que es ligeramente más largo que las pipetas de parche. </li><li> Tire de pipetas de parche en un extractor programable de tubo de vidrio de borosilicato con un filamento interno. Fabricación las micropipetas el día del experimento y utilizarlos inmediatamente. NOTA: Para las ZEC de grabación, se utiliza la siguiente configuración de tirador: calor: 484; tire: 0; velocidad: 25; el tiempo de retardo: 1; presión: 400 y un valor de rampa de 462. El OD e ID de los tubos de borosilicato son 1,65 mm y 1,0 mm, respectivamente. Resistencia de las pipetas es 10-14 mO. </li><li> Una hora antes de la cosecha de tejidos, descongelar un tubo de solución interna por Shaking en un vórtice para&gt; 30 min. </li></ol> 3. La disección de la retina <ol><li> Anestesiar al animal en un 4% de isoflurano en oxígeno hasta que el animal pierde la capacidad de respuesta a una pizca dedo del pie. Sacrificar al animal por dislocación cervical. </li><li> Corte ambos globos oculares fuera de los nervios ópticos a 1-2 mm de distancia de las cabezas del nervio óptico con Iris-rectas señalado tijeras. </li><li> Rollo de los globos oculares en una toalla de papel limpia para eliminar la sangre y luego sumergirlos en una solución de Ringer carbogenated mamíferos. Haz un agujero en el limbo con una aguja de 23 G y la bisectriz de los globos oculares cortando a lo largo del limbo con micro-tijeras. Retire la córnea y el cristalino utilizando unas pinzas finas. <ol><li> Para todo el montaje de la retina, pelar suavemente toda la retina fuera del epitelio pigmentado con unas pinzas finas y hacer cortes cuatro 1,5-2 mm ortogonales desde el borde hacia la cabeza del nervio óptico. </li></ol></li><li> Sumergir una membrana de nitrocelulosa perforado en el plato y arrastrar suavemente sobre la retina conGCL el lado hacia arriba. Coloque la cabeza del nervio óptico en el orificio central en la preparación de todo el montaje en una retina. La transferencia de la membrana con la retina en otro plato limpio. aplanar suavemente la retina con un pincel fino para parecerse a una cruz de Malta y sentar los cuatro bordes a través de los agujeros. <ol><li> Si una fracción de la retina debe ser examinado en un momento, corte cada ocular preparado a partir de la Etapa 3.3 en 3-4 piezas con una cuchilla de afeitar con el epitelio pigmentado permanece unido. Proteger las piezas no utilizadas de la luz en la solución externa carbogenated y usarlos dentro de las 12 horas. </li></ol></li><li> Seque la membrana de nitrocelulosa con un trozo de papel de filtro seco y eliminar la membrana limitante interna vítreo y con unas pinzas y un pincel. </li><li> Asegúrese de que todos los bordes de la retina están totalmente unidos a la membrana antes de transferir el conjunto en la cámara de grabación con un cubreobjetos de vidrio sellado en la parte inferior. Asegure la membrana con grasa de vacío a la hoja de la cubierta y rehydrate la retina con la solución externa. Tenga cuidado no para atrapar las burbujas de aire debajo de la asamblea. </li><li> Ajuste la cámara en el escenario de un microscopio vertical. Perfundir la cámara con (34-35 ° C) solución externa caliente carbogenated a una velocidad de ~ 3 ml por min. </li><li> Examinar la retina bajo una lente objetivo de 10x primero y, a continuación, utilizar una lente de 60x de inmersión en agua para ver neuronas GCL y INL bajo contraste de interferencia diferencial (DIC) y / o de epifluorescencia. Para visualizar las ZEC tdTomato expresan, utilice una fuente de luz blanca LED en combinación con un filtro de excitación de 554 nm y un filtro de emisión de 581 nm. </li></ol> 4.-Patch Clamp células enteras de grabación del plano de montaje en la retina <ol><li> Filtrar la solución interna a través de un filtro de jeringa en el filamento relleno personalizado. </li><li> Inserte el filamento en una micropipeta recién tirada y dispensar la solución interna cerca de la punta hasta que la solución cubre el electrodo de alambre de plata para&gt;5 mm. Sujetar la micropipeta sobre un soporte del electrodo con un poste de succión, a través del cual la presión dentro del electrodo se puede ajustar empujando o tirando el émbolo de una jeringa de plástico de 10 ml de tubo conectado. </li><li> Encuentra la pipeta con arreglo al objetivo y llevarlo hasta ~ 100 m por encima de la retina. Bajo el modo actual seguidor (I = 0), el uso DC offset a cero la señal de tensión de corriente continua de pie. Mida la resistencia de la pipeta bajo la grapa de corriente en modo (I Clamp) mediante la inyección de corrientes de onda cuadrada de amplitud fija a través de la pipeta mientras está en el baño y neutralizar la diferencia girando el mando rAccede. Utilice la lectura en el pomo rAccede para calcular la resistencia de la pipeta por la ley de Ohm. </li><li> Poco a poco llevar el electrodo de ~ 10 m por encima de la retina. Aplicar presión positiva al electrodo. Ver el cambio de la reflexión cerca de la punta de la pipeta cuando se aproxima a la retina. Rápidamente, pero forzar suavemente la pipeta en el GCL y reducir el pressu positivovolver inmediatamente. </li><li> Mover la pipeta hacia una neurona marcada. Evitar el contacto con otras neuronas, vasos sanguíneos y endfeet de células de Muller. Aplique presión más positiva si es necesario para evitar la obstrucción del electrodo. </li><li> Coloque la punta de la pipeta cerca de la línea media de una neurona marcada hasta un hoyuelo es visible. Liberar la presión positiva y permitir que la membrana plasmática para rebotar de nuevo en la punta de la pipeta. </li><li> Aplicar 20 a 120 pA corrientes negativas a la pipeta para ayudar a la formación de un sello giga-ohm. Aplicar una succión suave si es necesario tirar de la membrana plasmática en la pipeta. Esperar 5 min después de la formación del sello a la ruptura de la membrana celular. Esto permite que la solución interna derramado a que se solucione mediante superfusión. Romper la membrana por una suave succión. </li><li> Después de la ruptura de la membrana y en el modo de fijación de corriente, cambiar el equilibrio del puente y ajustar con el mando rAccede. NOTA: Para una pequeña celda como el SAC, sólo es necesario un ligero ajuste, en su caso. Alterntivamente, ficha excitatoria y inhibitoria postsináptica corrientes en el modo de fijación de voltaje (V Clamp) por la celebración de la célula en los potenciales de inversión de cloruro (alrededor de -75 mV) y glutamato (en torno a 0 mV), respectivamente. Comprobar y ajustar la posición de la pipeta si es necesario para dar cabida a la retina ocasional y / o la deriva micromanipulador. </li><li> Para grabar el potencial de membrana o cambio de corriente antes, durante y después del tratamiento farmacológico, preparar bloqueadores sinápticas (por ejemplo, CNQX, AP5, picrotoxina, tubocurarina, etc.) y moduladores del canal (por ejemplo, flupirtina, dopamina, ácido meclofenámico, etc.) fresca en el día del experimento de las existencias congeladas. Diluir los fármacos con solución de Ringer carbogenated. Aplicarlos de manera lotes a través de la perfusión. </li><li> Después de la grabación, eliminar suavemente la pipeta del soma. Transferir el conjunto de la cámara a un plato limpio. Desconecte y vuelva a colocar la retina en otra membrana de nitrocelulosa aplanado y sin hol perforadaes para asegurar la planitud retina durante la fijación. </li><li> Fijar la retina por inmersión en paraformaldehído al 4% durante 30 min a RT. Retire la retina de la membrana de nitrocelulosa y enjuagarlo extensamente con PBS 1x. Revelar la morfología de las células registradas por O tinción / N con estreptavidina conjugada con colorante diluido en 1x PBS con 0,4% de Triton X-100 a 4 ° C 14. Montar la retina en un medio antifade y observar las células grabados utilizando un microscopio confocal de barrido. NOTA: Los procedimientos que implican paraformaldehído, que es volátil y tóxico, deben llevarse a cabo en un proyecto de la cámara de seguridad. </li></ol>

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The authors have nothing to disclose.

Muchos laboratorios han registrado desde las neuronas GCL en la preparación 15-18 plana de montaje, pero nuestros procedimientos permiten la grabación de las neuronas de la INL. Por la presente hacemos hincapié en varios pasos que son críticos para las grabaciones de rutina con éxito. La frescura y planeidad de la retina son importantes para penetrar en él con una pipeta de grabación. En este sentido, la firma de la unión de la retina a la membrana de nitrocelulosa perfora...

As an easily accessible part of the central nervous system, the retina has for decades been a useful model in neuroscience studies. Genetic marking of neurons has allowed detailed characterization of synaptic connections in the retina. With many methodologies available to examine function and morphology of retinal neurons, the patch clamp recording technique has been instrumental in our current understanding of vertically transmitted signals in the retina. These signals are originated from photon absorption in photoreceptors and sent to brain visual centers through spiking of retinal ganglion cells (RGCs). Despite a large body of knowledge accumulated thus far, neural diversity in vascularized mammalian retina remains unsolved and obstructs the full appreciation of retinal circuits that subserve normal vision. This is in part because most recordings were performed on retinal slices to trade lateral circuit integrity for access to more proximal retinal neurons1-3. To gain a comprehensive picture on how retina computes visual signals, it is thus desirable to record neurons in flat-mounts wherein lateral connections, large and small, may be better preserved.
When synaptic transmission from photoreceptors to bipolar cells is interrupted due to a defective metabotropic glutamate receptor 6 (mGluR6) signaling pathway in depolarizing bipolar cells4-6 or simply as the result of photoreceptor loss in degenerated retinas7-10, many RGCs exhibit oscillatory activities. These oscillations originate from multiple sources, however the one involving gap junction coupling between AII amacrine cells (AII-ACs) and depolarizing cone bipolar cells (DCBCs) has received the most attention and hence is best understood1,7,11. We have found another source, which persists under pharmacological blockade of the aforementioned AII-AC/DCBC network and drives oscillation of OFF-type SACs in Rho&#916;CTA and Nob mice with deafferentated retinas7,8,12. Here we detail our protocol of preparing retinal flat-mounts for INL neuron recording. This approach uses commercial mouse lines (Jax stock no. 006410 and 007905) to mark cholinergic retinal neurons by fluorescent protein (tdTomato) expression that is identifiable under a fluorescent microscope equipped with contrast enhancing optics. Some experimental results acquired through this approach have been previously reported4,5,7,13.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Fixed-stage fluorescent microscope with DIC</td>
			<td style="width:155px;">Olympus</td>
			<td style="width:219px;">BX51-WI</td>
			<td style="width:419px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Micromanipulators</td>
			<td style="width:155px;">Sutter</td>
			<td style="width:219px;">MP-225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Patch clamp amplifier</td>
			<td style="width:155px;">A-M System</td>
			<td style="width:219px;">AM2400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">AD converter</td>
			<td style="width:155px;">National Instrument</td>
			<td style="width:219px;">NI-USB-6221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Heater controller</td>
			<td style="width:155px;">Warner Instrument</td>
			<td style="width:219px;">TC-324B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Inline heater</td>
			<td style="width:155px;">Warner Instrument</td>
			<td style="width:219px;">SC-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Peristaltic pump</td>
			<td style="width:155px;">Rainin</td>
			<td style="width:219px;">Dynamax</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">pipette puller</td>
			<td style="width:155px;">Sutter Instrument</td>
			<td style="width:219px;">P-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Glass tube with filament</td>
			<td style="width:155px;">King Precision Glass</td>
			<td style="width:219px;">Customized</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Stimulator</td>
			<td style="width:155px;">A.M.P.I.</td>
			<td style="width:219px;">Master-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Biocytin</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">B4261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">NaCl</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">S6191</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">KCl</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">P5405</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaHCO3</td>
			<td style="width:155px;">Fisher</td>
			<td style="width:219px;">BP328-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Na2HPO4</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">S0876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">NaH2PO4</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">S5011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">CaCl2</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">C5670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">MgSO4</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">M1880</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">D-glucose</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">G6152</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">K-gluconate</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">G4500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">ATP-Mg</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">A9187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Li-GTP</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">G5884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">EGTA</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">E0396</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">HEPES</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">H4034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">KOH</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">P5958</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Cs-methanesulfonate</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:219px;">C1426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">CsOH</td>
			<td style="width:155px;">Sigma</td>
			<td align="right" style="width:219px;">232041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Syringer filter</td>
			<td style="width:155px;">Nalgene</td>
			<td align="right" style="width:219px;">171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">1 ml syring</td>
			<td style="width:155px;">Rainin</td>
			<td align="right" style="width:219px;">17013002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">10 ul pipette tip</td>
			<td style="width:155px;">Genesee Scientific</td>
			<td style="width:219px;">24-130RL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Streptavidin-488</td>
			<td style="width:155px;">ThermoFisher</td>
			<td style="width:219px;">S-11223</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">10X PBS</td>
			<td style="width:155px;">Lonza</td>
			<td style="width:219px;">17-517Q</td>
			<td></td>
		</tr>
	</tbody>
</table>

patch clamp recording of starburst amacrine cells in a flat-mount preparation of deafferentated mouse retina

La retina de mamífero es un tejido en capas compuesta de múltiples tipos neuronales. Para entender cómo las señales visuales se procesa dentro de su red sináptica intrincada, registros electrofisiológicos se utilizan con frecuencia para estudiar las conexiones entre las neuronas individuales. Hemos optimizado un preparado de montaje plana para la grabación de patch clamp de neuronas marcadas genéticamente en tanto GCL (capa de células ganglionares) y INL (capa nuclear interna) de las retinas de ratones. Grabación de las neuronas en INL-montajes planos se ve favorecida sobre las rebanadas porque las conexiones verticales y laterales se conservan en la antigua configuración, permitiendo que los circuitos de la retina con grandes componentes laterales para ser estudiado. Hemos utilizado este procedimiento para comparar las respuestas de las neuronas espejo, mediante asociación en las retinas como las células amacrinas starburst colinérgicos (ZEC).

Grabaciones representativas de dentro y fuera de tipo ZEC de una retina del ratón deafferentated se muestran en la Figura 1. Células colinérgicas en tanto GCL y INL se pueden identificar de forma fiable mediante fluorescencia tdTomato y específicas para la grabación de células enteras patch clamp bajo DIC (Figura 1A) para revelar la oscilación de sus potenciales de membrana (Top trazas) y las corrientes sinápticas que la impulsan (trazas de fondo...

Watch this Scientific Journal Video about Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina at JoVE.com

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

Este protocolo muestra cómo realizar la grabación de células enteras patch clamp en neuronas de la retina de una preparación plana de montaje.

Patch Clamp grabación de células amacrinas Starburst en un plano de montaje Preparación de Deafferentated retina del ratón

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

patch-clamp-recording-starburst-amacrine-cells-flat-mount-preparation

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

8.1K vistas.  Baylor College of Medicine.  Este protocolo muestra cómo realizar la grabación de células enteras patch clamp en neuronas de la retina de una preparación plana de montaje. 

Video: Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

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

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike 1. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex 2. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative 3, 4: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening 5. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons 6; recently, we have also reported progress towards interrogating mammalian neurons 7. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel.The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
<img alt="Figure 1" src="/files/ftp_upload/3288/3288fig1.jpg" />
Figure 1. Principle of measurement using the NRCC patch-clamp chip
We detail here the protocols to sterilize and prime the chips, load them with medium, plate them with cells, and finally use them for electrophysiological recordings.

We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings.

El cultivo y la electrofisiología de las células en NRCC de patch-clamp chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

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 ...

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.

Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.

Whole Cell Patch Clamp para investigar los mecanismos de la Estimulación Neural infrarrojos

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

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 ...

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. ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Perforado grabación de patch-clamp de ratón neuronas sensoriales olfativas en neuroepitelio Intacto: Análisis funcional de las neuronas que expresan un odorante del receptor Identificado

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

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 ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

Preparación de cortes horizontales de ratón adulto Retina de Estudios electrofisiológicos

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...