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.

A aprovação ética - procedimentos que envolvem indivíduos animais foram conduzidos de acordo com as regras e regulamentos dos Institutos Nacionais de diretrizes de saúde para animais de pesquisa, conforme aprovado pelo Institutional Animal Care e usar comitê de Baylor College of Medicine. 1. externa e Soluções Internas <ol><li> Usar a solução de Ringer de mamífero durante a dissecção retina e como a solução externa na gravação eletrofisiológico subsequente. Preparar a solução de Ringer de mamífero a partir de solução-mãe 10x (sem cálcio) no dia da gravação, e adicionar CaCl2 gota a gota, após 15 minutos de carbogenation (95% O 2 e 5% CO 2). A solução final 1x contém (em mM): 120 NaCl, 5 KCl, 25 NaHCO3, 0,8 Na 2 HPO 4, 0,1 NaH 2 PO 4, 2 CaCl2, 1 MgSO4 e 10 de D-glucose. </li><li> Use duas soluções internas para caracterizar oscilações SAC. Testea utilidade das soluções preparadas em de célula inteira gravações patch clamp prolongados em RGCs rato adulto e armazenar os lotes verificados em alíquotas de 500 uL a -20 ° C até à sua utilização. <ol><li> Para a gravação de membrana oscilação potencial, usar uma solução interna à base de potássio que contém (em mM): 125 de K-gluconato, 8 de NaCl, 4 de ATP-Mg, 0,5 Li-GTP, 5 EGTA, 10 HEPES e 0,2% biocitina (w / v). Ajustar o pH para 7,3 com KOH. </li><li> Para gravação excitatórios e correntes pós-sinápticos de inibição, usar uma solução interna à base de césio que contém (em mM): 100 Cs-metanossulfonato, 8 de NaCl, 4 de ATP-Mg, 0,5 Li-GTP, 5 EGTA, 10 HEPES e 0,2% biocitina ( w / v). Ajustar o pH para 7,3 com CsOH. </li></ol></li></ol> 2. Preparação para o Dia da Gravação <ol><li> Para preparar uma membrana de nitrocelulose com buracos abertos, perfurar manualmente a membrana com um perfurador personalizado feito de uma agulha de seringa embotada 16 G e achatar a membrana entre duas placas de vidro limpo. Para uma whole-montar retina, faça um furo central de 0,2 mm de diâmetro e cercá-lo por 4 furos maiores que são 1-1,2 mm de diâmetro. </li><li> Para fazer um filamento aterro personalizado para carregar solução interna para eletrodos, derreta uma ponta de pipeta 10 ul no meio e gentilmente alongar a parte derretida até que esfria e endurece. Imediatamente antes da utilização, usar uma lâmina de barbear para cortar limpo do filamento até que é ligeiramente mais longo do que as pipetas de patch. </li><li> Puxe pipetas de patch em um extrator programável de tubo de vidro de borosilicato com um filamento interno. Fabricar as micropipetas no dia da experiência e usá-los imediatamente. NOTA: Para ZEC gravação, usamos a seguinte configuração extrator: calor: 484; puxe: 0; velocidade: 25; tempo de atraso: 1; pressão: 400 e um valor de rampa de 462. O OD e ID dos tubos de borosilicato são de 1,65 mm e 1,0 mm, respectivamente. Resistência das pipetas é 10-14 mohms. </li><li> Uma hora antes da colheita de tecidos, descongelar um tubo de solução interna por shaking-lo em um vórtice de&gt; 30 min. </li></ol> 3. Retina Dissection <ol><li> Anestesiar o animal por 4% de isoflurano em oxigênio até que o animal perde a capacidade de resposta a uma pitada dedo do pé. Sacrificar o animal por deslocamento cervical. </li><li> Corte ambos os globos oculares fora os nervos ópticos a 1-2 mm de distância das cabeças do nervo óptico com íris retas-apontou tesoura. </li><li> Rolar os olhos em uma toalha de papel limpa para remover sangue e, em seguida, mergulhe-os em solução de Ringer de mamífero carbogenated. Faça um furo no limbo com uma agulha G 23 e bissetriz os globos oculares cortando ao longo do limbo com micro-tesoura. Retirar a córnea ea lente usando uma pinça fina. <ol><li> Para retinal todo-mount, descascar delicadamente a retina inteira fora do epitélio pigmentado com uma pinça fina e fazer cortes quatro 1,5-2 mm ortogonais a partir da borda em direção à cabeça do nervo óptico. </li></ol></li><li> Mergulhe uma membrana de nitrocelulose soco no prato e gentilmente arraste a retina sobre ele como lado GCL-se. Coloque a cabeça do nervo óptico no orifício central na preparação de uma retina de toda a montagem. Transferir a membrana com a retina no outro prato limpo. Gentilmente achatar a retina com um pincel fino para olhar como uma cruz de Malta e colocar todas as quatro bordas ao longo dos buracos. <ol><li> Se uma fracção da retina é para ser examinado de cada vez, cortar cada ocular preparado a partir do Passo 3.3 em 3-4 pedaços com uma lâmina de barbear com o epitélio pigmentado permanece ligado. Proteger as peças não utilizadas de luz na solução externa carbogenated e usá-los dentro de 12 horas. </li></ol></li><li> Seque a membrana de nitrocelulose com um pedaço de papel de filtro seco e remover a membrana limitadora interna e vítreo com uma pinça e um pincel. </li><li> Certifique-se de que todas as bordas da retina são totalmente ligado à membrana antes de transferir a montagem para a câmara de gravação com uma lamela de vidro selado na parte inferior. Fixe a membrana com graxa de vácuo para a lamínula e rehydratE é a retina com a solução externa. Seja não tomar cuidado para prender as bolhas de ar debaixo da montagem. </li><li> Definir a câmara para o palco de um microscópio vertical. Perfundir a câmara com (34-35 ° C) solução externa carbogenated quente, a uma taxa de ~ 3 mL por min. </li><li> Examinar a retina sob uma lente objetiva de 10x em primeiro lugar e, em seguida, usar uma lente de 60x de imersão em água para ver neurônios GCL e INL sob contraste de interferência diferencial (DIC) e / ou de epifluorescência. Para visualizar ZEC-expressando tdTomato, utilizar uma fonte de luz LED branco em combinação com um filtro de excitação de 554 nm e um filtro de emissão de 581 nm. </li></ol> 4. Whole-cell patch de gravação braçadeira de Flat-mount Retina <ol><li> Filtrar a solução através de um filtro interno da seringa no filamento aterro costume. </li><li> Insira o filamento em uma micropipeta acabada de tirar e dispensar a solução interna perto da ponta até que a solução cobre o fio do eletrodo de prata para&gt;5 mm. Fixar a micropipeta para um suporte de eléctrodo com uma vara de aspiração, através da qual a pressão no interior do eléctrodo pode ser ajustada, empurrando ou puxando o êmbolo de uma seringa de plástico de 10 ml ligado de tubo. </li><li> Encontre a pipeta no âmbito do objectivo e trazê-lo para baixo para ~ 100 mm acima da retina. Sob o modo atual seguidor (I = 0), use DC offset para zero o sinal de tensão DC em pé. Meça a resistência da pipeta sob a pinça de corrente de modo (I Grampo) através da injeção de amplitude fixa correntes onda quadrada através da pipeta enquanto ele está no banho e neutralizar a diferença girando o botão rAcesse. Use a leitura na maçaneta rAcesse para calcular a resistência da pipeta pela lei de Ohm. </li><li> levar lentamente o eletrodo para ~ 10 mm acima da retina. Aplicar a pressão positiva para o eléctrodo. Assista a mudança reflexão perto da ponta da pipeta quando se aproxima da retina. Rapidamente, mas com cuidado forçar a pipeta no GCL e reduzir o pressu positivare imediatamente. </li><li> Mover a pipeta para um neurônio rotulados. Evite entrar em contato com outros neurónios, vasos sanguíneos e endfeet de células Muller. Aplique uma pressão mais positiva se for necessário para evitar o entupimento do eletrodo. </li><li> Posicione a ponta da pipeta perto da linha média de um neurônio rotulados até uma covinha é visível. Solte a pressão positiva e permitir que a membrana plasmática para saltar de volta para a ponta da pipeta. </li><li> Aplicar 20 a 120 pA correntes negativos para a pipeta para ajudar a formação de um selo giga-ohm. Aplicar uma sucção suave, se necessário, para puxar a membrana plasmática para a pipeta. Esperar 5 minutos após a formação de selo para romper a membrana celular. Isso permite que a solução interna derramado foi afastada pelo superfusão. Ruptura da membrana por uma sucção suave. </li><li> Após a ruptura da membrana e ao mesmo tempo no modo de pinça de corrente, ligue o equilíbrio ponte e ajustá-lo usando o botão rAcesse. NOTA: Por uma pequena célula como o SAC, só é necessário ligeiro ajuste, se houver. Alterntivamente, ficha excitatórios e inibitórios correntes pós-sinápticos no modo braçadeira de tensão (V Grampo) por segurando o celular no potencial de reversão de cloreto de (cerca de -75 mV) e glutamato (em torno de 0 mV), respectivamente. Verifique e ajuste a posição da pipeta se necessário para acomodar retina ocasional e / ou deriva micromanipulador. </li><li> Para registar o potencial de membrana ou alteração da corrente antes, durante e após o tratamento farmacológico, preparam sinápticas bloqueadores (por exemplo, CNQX, AP5, picrotoxina, tubocurarina, etc.) e moduladores do canal (por exemplo, flupirtina, dopamina, ácido meclofenâmico, etc.) em fresco o dia do experimento de stocks congelados. Diluir as drogas com solução carbogenated de Ringer. Aplicá-las de uma forma de lote por meio de perfusão. </li><li> Após a gravação, remover suavemente a pipeta da soma. Transferir a montagem da câmara para um prato limpo. Desligar e voltar a ligar a retina para outra membrana de nitrocelulose achatados sem hol perfuradoES para assegurar o nivelamento da retina durante a fixação. </li><li> Fixar a retina por imersão em 4% de paraformaldeído durante 30 min à TA. Remova a retina a partir da membrana de nitrocelulose e lave-o extensivamente com 1x PBS. Revelam a morfologia das células gravadas por O / N coloração com estreptavidina conjugado com corante diluídos em 1x PBS com 0,4% de Triton X-100 a 4 ° C 14. Monte a retina em meio Antifade e observar células gravado utilizando um microscópio de varredura confocal. NOTA: os procedimentos de paraformaldeído, que é volátil e tóxico, deve ser realizado em um projeto de câmara de segurança. </li></ol>

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The authors have nothing to disclose.

Muitos laboratórios têm gravado a partir de neurônios GCL na preparação 15-18 plana de montagem, mas os nossos procedimentos de permitir a gravação de neurônios do INL. Vimos por este meio enfatizar várias etapas que são críticas para as gravações de rotina bem sucedidas. A frescura e achatamento da retina são importantes para penetrar com uma pipeta de gravação. A este respeito, a ligação firme da retina para a membrana de nitrocelulose perfurado é primordial e...

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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	</colgroup>
	<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

A retina dos mamíferos é um tecido em camadas compostas de vários tipos neuronais. Para entender como os sinais visuais são processados ​​dentro de sua rede sináptica intrincado, registros eletrofisiológicos são frequentemente usados ​​para estudar as conexões entre os neurônios individuais. Nós otimizamos uma preparação plana de montagem para a gravação de patch-clamp de neurônios geneticamente marcados em ambos GCL (camada de células ganglionares) e INL (camada nuclear interna) de retinas de ratos. Gravação de neurônios do INL em planos montagens é favorecido sobre fatias porque as conexões verticais e laterais são preservados na configuração anterior, permitindo que os circuitos da retina com grandes componentes laterais a ser estudado. Temos utilizado este procedimento para comparar as respostas dos neurônios-espelho-partnered em retinas tais como as células amácrinas starburst colinérgicos (ZEC).

Registos representativos de dentro e fora do tipo SACs de uma retina deafferentated rato são mostrados na Figura 1. As células colinérgicas em ambos GCL e INL podem ser identificados de forma confiável por tdTomato fluorescência e direcionados para de células inteiras de gravação patch-clamp sob DIC (Figura 1A) para revelar oscilação de seus potenciais de membrana (top traços) e as correntes sinápticas que dirigi-lo (traços inferiores, <stro...

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 demonstra como executar de células inteiras de gravação patch-clamp em neurônios da retina a partir de uma preparação flat-mount.

Patch-clamp gravação de células Starburst Amácrinas em um monte de Luxo Preparação de Deafferentated mouse Retina

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

Research

JoVE Journal

Neuroscience

8.1K visualizações.  Baylor College of Medicine.  Este protocolo demonstra como executar de células inteiras de gravação patch-clamp em neurônios da retina a partir de uma preparação flat-mount. 

Vídeo: 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.

Remendo Recordings Grampo de neurônios da retina do rato em um Preparação Slice Dark-adaptados

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

Neurociência

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.

Cultura e Eletrofisiologia de células em 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 ...

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.

Gravações patch clamp em células ciliadas da orelha interna Isolado de 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  ...

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; Ó</sub&gt; ATPase Vesicle Preparação e Técnica para Execução de Remendo Recordings Grampo de membranas de vesículas submitocondriais

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.

Todo patch clamp celular para investigar os mecanismos de estimulação neural Infrared

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.

Grampo patch Gravações de Embryonic Cells Zebrafish 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.

Gravação Light-provocou respostas pós-sinápticos nos neurônios em Dark-adaptados, Preparações da retina do rato fatia usando técnicas Grampo patch

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.

Perfurado gravação patch-clamp de rato Olfactory neurônios sensoriais na Intact neuroepitélio: Análise Funcional do Neurônios Expressando uma Odorant Receptor Identificados

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.

Gravações de células inteiras de patch-clamp em 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 ...

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.

Preparação de horizontais Fatias de rato adulto Retina de Estudos eletrofisiológicos

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