Agradecemos a Maila Reh, Tharsana Tharmalingam e especialmente a Altina Klein pela excelente assistência técnica. Este trabalho foi apoiado pela Fundação de Pesquisa Alemã (DFG) (SFB1078 B2, FOR1279 SPP1665 a PH) e o Cluster of Excellence Unifying Concepts em Catálise, UniCat, BIG-NSE (JV) e E4 (PH).

<ol>
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The authors have nothing to disclose.

A determinação de potenciais de reversão em condições iónicas e elétricas definidas fornece informações sobre a espécie de íons transportada após a ativação leve de ChRs. Se exclusivamente uma espécie de iões é variada num meio fisiológico complexo e o potencial de reversão obtido se desloque de acordo com o potencial teórico de Nernst, esta espécie de iões é a única transportada. No entanto, para ChRs, reversão potenciais turnos são geralmente menos pronunciada do que o esperado a partir da equação de Nernst devido à permeabilidade para diferentes espécies iónicas, bem como a concorrência entre íons conduzidos. Neste caso, a situação torna-se mais complexa e todos os íons potencialmente conduzidos devem ser trocados separadamente exigindo uma variedade de soluções de medição. Além disso, a maioria dos CCRs e as primeiras ACRs 9 , 10 , 21 artificiais são condutoras para protões mas concentrações de protões variáveisA concentração de protões pode não só alterar os potenciais de reversão, como também influenciar a condutância iónica 9 , a cinética das fotocorriças 21 , 36 e a sensibilidade à luz espectral 11 , 37 , devido à alteração das redes de ligação de hidrogénio, ao estado de protonação de Resíduos individuais e alterações estruturais secundárias. A condutï¿½cia do protï¿½ pode ser direccionada por reduï¿½o das concentraï¿½es de todos os outros iï¿½es potencialmente conduzidos tais como Na + , Ca 2+ ou Cl - mantendo o pH constante para evitar as alteraï¿½es de proteï¿½as mencionadas acima. Substitutos não conduzidos são geralmente moléculas carregadas e carregadas como NMG + para os catiões 12 , e gluconato, 2- ( N- morfolino) etanossulfonato (MES) ou aspartato para aniões. Comparando potenciais de reversão em diferentes iPermite então o cálculo da permeabilidade iónica relativa em condições de equilíbrio pela equação de tensão de Goldman-Hodgkin-Katz. Quantificar a contribuição exata de diferentes íons para fotocorrentes líquidas, considerando a competição de íons além das condições de equilíbrio, exige modelos matemáticos complexos 12 , 38 . Conhecer a composição de íons transportados por ChRs ajuda a elucidar possíveis efeitos colaterais resultantes da aplicação de ChR especialmente em um contexto neurofisiológico. Por exemplo, a activação prolongada de ChR pode causar acidificação intracelular 39 ou condução de Ca2 + pode desencadear a libertação sináptica e as vias de segundo mensageiro 40 . Uma vez que as células são habitualmente empacotadas dentro dos tecidos, a activação das rodopsinas microbianas pode não apenas influenciar a composição do ião intracelular, mas também a luz extracelular, afectando assim nCélulas vizinhas 41 . Apesar da seletividade iónica, outras características biofísicas de ChRs como amplitudes fotocorrentes, inativação, sensibilidade espectral ou de luz e propriedades cinéticas são freqüentemente de interesse para conduzir, projetar e interpretar experimentos envolvendo técnicas optogenéticas. Algumas destas propriedades também podem ser determinadas a partir do protocolo acima como descrito em qualquer outra parte 18 , 37 , 42 . Para investigar a sensibilidade à luz da célula de expressão de ChR, a intensidade de luz pode ser variada ajustando a potência da fonte de luz ou atenuando a luz de activação com filtros de densidade neutra. Para obter a sensibilidade espectral, é necessária uma fonte de luz policromática e as intensidades têm de ser ajustadas para o fluxo de fótons e largura de banda iguais para todos os comprimentos de onda. Os espectros de alta intensidade apenas se assemelham aos espectros de absorção do fotorreceptor se pulsos de luz curtos ouSão utilizados flashes de laser, caso contrário podem ocorrer fenómenos de adaptação e reacções fotoquímicas de retorno. A recuperação de ChR parcialmente activado para ChR totalmente activo (tempo total de renovação de fotociclo) pode ser sondada aplicando um protocolo de duplo impulso com intervalos escuros crescentes entre os dois impulsos de luz. As propriedades biofísicas de ChRs discutidas acima são importantes para a escolha de um ChR adequado combinando as necessidades experimentais 40 . Derivar estas propriedades biofísicas por técnicas eletrofisiológicas diretamente se assemelha a função de proteína que é mal coberto por métodos espectroscópicos. Após o estabelecimento bem sucedido de medições de tensão-braçadeira, a técnica pode ser combinada com abordagens de engenharia de proteínas como a mutagénese dirigida 9 , 11 , 43 , 44 , a mistura de hélice 37 , <supClass = &quot;xref&quot;&gt; 45, introdução de seqüências de direcionamento 46 ou fusão com outras proteínas 41 , 47 . Isso aumentou o conhecimento molecular sobre ChRs e seu modo de operação e criou uma grande diversidade de variantes ChR com cinética alterada, condutância, sensibilidade espectral e seletividade iónica 40 .

Channelrhodopsins (ChR) são luz-gated canais iônicos que ocorrem na mancha do olho de algas verdes motile, e servem como fotossensores primários para phototaxis e respostas fóbicas [ 1] . Desde sua primeira descrição em 2002 2 , os CHRs abriram o caminho para o campo emergente da optogenética e podem ser aplicados em uma variedade de células excitáveis, por exemplo, dentro dos músculos esqueléticos, do coração ou do cérebro 3 , 4 , 5 . A expressão de ChRs em células alvo resulta na permeabilidade iónica controlável pela luz da respectiva célula. Num contexto neuronal, isto permite a activação 6 , 7 , 8 ou inibição 9 , 10 de potencial de acção (AP) disparando - dependendo do ião conduzido - com o espaço e temporalPrecisão da luz enfatizando como a seletividade iónica de uma variante ChR determina sua aplicação optogenética. Os primeiros ChRs descobertos de Chlamydomonas reinhardtii e Volvox carteri são permeáveis ​​aos prótons, mas também aos catiões monovalentes como o sódio, o potássio e, em menor grau, os catiões divalentes como o cálcio eo magnésio 11 , 12 , 13 . Actualmente, mais do que 70 canais de canal condutores naturais (CCR) 14 , 15 , 16 , 17 e várias variantes de engenharia 18 , 19 , 20 com propriedades diferentes tais como Tamanho da fotocorriente, sensibilidade espectral, cinética e selectividade catiónica. Considerando que em neurociência, CCRs aRe usado para ativar as células e disparar APs, bombas microbianas conduzidas pela luz foram os únicos antagonistas disponíveis para silenciar neurônios por anos. Em 2014, dois grupos demonstraram simultaneamente que os CCRs podem ser convertidos em canalrhodopsins condutores de aniões (ACRs) por alteração da polaridade ao longo do suposto poro de condução iónica através da engenharia molecular 9 , 21 . Subseqüentemente, os ACRs naturais foram identificados em várias algas criptofitas 22 , 23 , 24 . Mais importante ainda, a ativação de luz de ACRs medeia as correntes de cloreto em neurônios adultos permitindo a inibição da atividade neuronal em intensidades de luz muito menores do que as bombas microbianas que apenas transportam cargas únicas por fóton absorvido. A actividade de ChR pode ser direccionada directamente por gravações electrofisiológicas de &quot;patch-clamp&quot; de correntes induzidas pela luz em células HEK293. O grampo de fixaçãoTécnica foi originalmente desenvolvida no final da década de 1970 25 e melhorada por Hamill et al. , Permitindo a gravação da entidade de correntes de uma célula pequena (modo célula inteira) com alta resolução de corrente e controle direto da tensão da membrana 26 . Aplicada em cultura de células, esta técnica proporciona um controlo preciso das condições de gravação iónicas e eléctricas e permite estudar a selectividade dos iões juntamente com a contribuição relativa dos iões para a corrente total. Aqui, exemplificamos o exame da seletividade iónica para a canalrhodopsina condutora de ânions de Proteomonas sulcata ( Ps ACR1) 22 , 23 através do registro das relações tensão-corrente em várias concentrações de cloreto extracelular para provar alta condutância ao cloreto.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >HEK293 cells</td>
			<td >Sigma Aldrich</td>
			<td >85120602</td>
			<td >Human embryonic kidney cells</td>
		</tr>
		<tr >
			<td >Retinal</td>
			<td >Sigma Aldrich</td>
			<td>R2500</td>
			<td>all-trans retinal</td>
		</tr>
		<tr >
			<td >FuGENE HD</td>
			<td >Promega</td>
			<td>E2312</td>
			<td>Transfection reagent</td>
		</tr>
		<tr >
			<td >DMEM</td>
			<td >Biochrome</td>
			<td>FG 0445</td>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
		</tr>
		<tr >
			<td >Agarose</td>
			<td >Roth</td>
			<td>3810</td>
			<td>Agar bridges</td>
		</tr>
		<tr >
			<td >CaCl2</td>
			<td >Roth</td>
			<td>5239</td>
			<td>CaCl2 2H2O</td>
		</tr>
		<tr >
			<td >CsCl</td>
			<td >Biomol</td>
			<td>2452</td>
			<td></td>
		</tr>
		<tr >
			<td >EGTA</td>
			<td >Roth</td>
			<td>3054</td>
			<td></td>
		</tr>
		<tr >
			<td >FBS</td>
			<td >Biochrome</td>
			<td>S0615</td>
			<td>Cell culture</td>
		</tr>
		<tr >
			<td >Glucose</td>
			<td >Roth</td>
			<td>HN06</td>
			<td>D(+)-Glucose</td>
		</tr>
		<tr >
			<td >KCl</td>
			<td >Roth</td>
			<td>6781</td>
			<td></td>
		</tr>
		<tr >
			<td >MgCl2</td>
			<td >Roth</td>
			<td>2189</td>
			<td>MgCl2 6H2O</td>
		</tr>
		<tr >
			<td >NaCl</td>
			<td >Roth</td>
			<td>3957</td>
			<td></td>
		</tr>
		<tr >
			<td >NMG</td>
			<td >Sigma Aldrich</td>
			<td>M2004</td>
			<td>N-Methyl-D-glucamine</td>
		</tr>
		<tr >
			<td >Na-Aspartate</td>
			<td >Sigma Aldrich</td>
			<td>A6683</td>
			<td>L-Aspartic acid sodium salt monohydrate</td>
		</tr>
		<tr >
			<td >Citric acid</td>
			<td >Roth</td>
			<td>6490</td>
			<td></td>
		</tr>
		<tr >
			<td >AgeI</td>
			<td >ThermoFischerScientific</td>
			<td>ER1462</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr >
			<td >XhoI</td>
			<td >ThermoFischerScientific</td>
			<td>ER0695</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr >
			<td >NheI</td>
			<td >ThermoFischerScientific</td>
			<td>ER0975</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr >
			<td >XL1Blue E.coli/</td>
			<td >Agilent Technologies</td>
			<td>200249</td>
			<td>Chemocompetent E.coli</td>
		</tr>
		<tr >
			<td >Kanamycin</td>
			<td >Roth</td>
			<td>T832</td>
			<td></td>
		</tr>
		<tr >
			<td >Lysogeny broth medium</td>
			<td >Roth</td>
			<td>X964</td>
			<td></td>
		</tr>
		<tr >
			<td >Agar-Agar</td>
			<td >Roth</td>
			<td>6494</td>
			<td>Agar plates</td>
		</tr>
		<tr >
			<td >Plasmid purification kit</td>
			<td >Marchery-Nagel</td>
			<td>740727.25</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicilin/Streptomycin</td>
			<td >Biochrome</td>
			<td>A 2213</td>
			<td>Cell culture</td>
		</tr>
		<tr >
			<td >Poly-D-lysine hydrobromide</td>
			<td >Sigma Aldrich</td>
			<td>P6407-5MG</td>
			<td>Cover slip coating</td>
		</tr>
		<tr >
			<td >Microforge</td>
			<td >Custom made</td>
			<td></td>
			<td>Fire polishing</td>
		</tr>
		<tr >
			<td >Serological pipettes</td>
			<td >TPP</td>
			<td></td>
			<td>Different sizes</td>
		</tr>
		<tr >
			<td >Clean bench</td>
			<td >Kojair</td>
			<td>Biowizard SL130</td>
			<td></td>
		</tr>
		<tr >
			<td >Stirrer</td>
			<td >IKA</td>
			<td>RCT classic</td>
			<td></td>
		</tr>
		<tr >
			<td >Silver wire</td>
			<td >Science Products</td>
			<td>AG-T25; AG-T10</td>
			<td>Electrodes, 0.64 mm (bath); 0.25 mm (electrode)</td>
		</tr>
		<tr >
			<td >pH-meter</td>
			<td >Knick</td>
			<td>765 Calimetric</td>
			<td></td>
		</tr>
		<tr >
			<td >Osmometer</td>
			<td >Vogel</td>
			<td>OM 815</td>
			<td></td>
		</tr>
		<tr >
			<td >Microscope</td>
			<td >Carl Zeiss</td>
			<td>ID03</td>
			<td>Fire polishing</td>
		</tr>
		<tr >
			<td >CO2 incubator</td>
			<td >Binder</td>
			<td>CB150</td>
			<td></td>
		</tr>
		<tr >
			<td >Cell culture dishes</td>
			<td >TPP</td>
			<td>93040</td>
			<td>34 mm internal diameter</td>
		</tr>
		<tr >
			<td >Cover slips</td>
			<td >Roth</td>
			<td>P232</td>
			<td>15 mm diameter</td>
		</tr>
		<tr >
			<td >Thermometer</td>
			<td >R&#246;ssel Messtechnik</td>
			<td>MTM12</td>
			<td></td>
		</tr>
		<tr >
			<td >Beamsplitter</td>
			<td >Chroma</td>
			<td>21011</td>
			<td>90/10 transmission</td>
		</tr>
		<tr >
			<td >Pipette holder</td>
			<td>ALA Scientific Instruments</td>
			<td>PPH-1P-AXU-0-1.5</td>
			<td></td>
		</tr>
		<tr >
			<td >Headstage</td>
			<td >Molecular Devices</td>
			<td>CV203BU</td>
			<td></td>
		</tr>
		<tr >
			<td >Amplifier</td>
			<td >Molecular Devices</td>
			<td >AxoPatch200B</td>
			<td></td>
		</tr>
		<tr >
			<td >Digitizer</td>
			<td >Molecular Devices</td>
			<td >DigiData1400</td>
			<td>Digital analog converter</td>
		</tr>
		<tr >
			<td >Lightsource</td>
			<td >TILL Photonics</td>
			<td >Polychrome V</td>
			<td>Set to 540 nm full intensity</td>
		</tr>
		<tr >
			<td >Microscope</td>
			<td >Carl Zeiss</td>
			<td >Axiovert 100</td>
			<td></td>
		</tr>
		<tr >
			<td >Shutter</td>
			<td >Vincent Associates</td>
			<td >VS25</td>
			<td></td>
		</tr>
		<tr >
			<td >Shutter driver</td>
			<td >Vincent Associates</td>
			<td >VCM-D1</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass capilarries</td>
			<td >Warner Instruments</td>
			<td>G150F-3</td>
			<td >Boresilicate capillaries with fire polished ends OD 1.5 mm ID&#160; 0.86 mm</td>
		</tr>
		<tr >
			<td >Micropipette puller</td>
			<td >Sutter Instruments</td>
			<td >P1000</td>
			<td></td>
		</tr>
		<tr >
			<td >Bath handler</td>
			<td >Lorenz Messger&#228;tebau</td>
			<td >MPCU</td>
			<td></td>
		</tr>
		<tr >
			<td >Tripleband filterset</td>
			<td >Chroma</td>
			<td>69008</td>
			<td>Fluorescence filter&#160; ECFP/EYFP/mCherry&#160;</td>
		</tr>
		<tr >
			<td >CCD camera</td>
			<td >Watec</td>
			<td >Wat-221SCCD</td>
			<td></td>
		</tr>
		<tr >
			<td >Optometer</td>
			<td >Gigahertz Optik</td>
			<td >P9710</td>
			<td>Measure light intensities</td>
		</tr>
		<tr >
			<td >Objective</td>
			<td >Carl Zeiss</td>
			<td>421462-9900-000</td>
			<td >W Plan-Apochromat 40X/1.0 DIC</td>
		</tr>
		<tr >
			<td >Micromanipulator</td>
			<td >Scientifica</td>
			<td>PatchStar</td>
			<td></td>
		</tr>
		<tr >
			<td >Recording chamber</td>
			<td >Custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Power supply</td>
			<td >Manson</td>
			<td>HCS-3202</td>
			<td>Avoids electrical noise from microscope built-in power supply</td>
		</tr>
		<tr >
			<td >Vibration isolated table</td>
			<td >Newport</td>
			<td>M-VW-3636-OPT-01</td>
			<td></td>
		</tr>
		<tr >
			<td >Faraday cage</td>
			<td >Custom made or any commercial matching table</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Hoses</td>
			<td >Any comercial; e.g. Roth</td>
			<td></td>
			<td>Different sizes and materials for bath handling and application of pipette pressure; agar bridges</td>
		</tr>
		<tr >
			<td >Linear shaker</td>
			<td >Sunlab Instruments</td>
			<td>SU 1000</td>
			<td></td>
		</tr>
		<tr >
			<td >Liquid junction potential calculator</td>
			<td >Molecular Devices or directly from Peter H. Barry</td>
			<td></td>
			<td>Program is included in the Clampex aquisition software or can be obtained from&#160; p.barry@unsw.edu.au</td>
		</tr>
		<tr >
			<td >Data acquisition software</td>
			<td >Molecular Devices</td>
			<td>Clampex 10.X</td>
			<td></td>
		</tr>
		<tr >
			<td >Data evaluation software</td>
			<td >Molecular Devices</td>
			<td>Clampfit 10.X</td>
			<td></td>
		</tr>
		<tr >
			<td >PsACR1</td>
			<td>GenBank or Addgene</td>
			<td >KF992074.1 or Addgene plasmid #85465</td>
			<td>Gene encoding for PsACR1</td>
		</tr>
		<tr >
			<td >Amplifier guide</td>
			<td >Molecular Devices</td>
			<td>The Axon Guide</td>
			<td></td>
		</tr>
	</tbody>
</table>

whole-cell patch-clamp recordings for electrophysiological determination of ion selectivity in channelrhodopsins

Ao longo da última década, os channelrhodopsins tornaram-se indispensáveis ​​na pesquisa neurocientífica onde eles são usados ​​como ferramentas para manipular não-invasivamente processos elétricos em células-alvo. Neste contexto, a selectividade iónica de uma canalrhodopsina é de particular importância. Este artigo descreve a investigação da seletividade de cloretos para uma canalrhodopsina de Proteomonas sulcata, condutora de aniões, recentemente identificada, por meio de gravações electrofisiológicas em células HEK293. O procedimento experimental para a medição de fotocorrentes de luz-gated exige uma fonte de luz rápida comutável - idealmente monocromática - acoplada ao microscópio de uma instalação de patch-clamp, de outra forma convencional. Os procedimentos preparativos antes da experiência são descritos envolvendo a preparação de soluções tamponadas, considerações sobre potenciais de junção de líquidos, semeadura e transfecção de células e puxar de pipetas de remendo. A gravação real da relação tensão-correnteS para determinar os potenciais de reversão para diferentes concentrações de cloreto ocorre 24 h a 48 h após a transfecção. Finalmente, os dados eletrofisiológicos são analisados ​​com respeito a considerações teóricas de condução de cloreto.

<html> A Figura 2 mostra resultados representativos obtidos a partir de medições seguindo o protocolo descrito. Durante a iluminação com luz verde, Ps ACR1 apresenta uma corrente transitória rápida que decai rapidamente para um nível de corrente estacionária. Depois que a luz é desligada, as fotocorrentes decaem para zero em milissegundos ( Figura 2A ). A troca da concentração de cloreto extracelular provoca um deslocamento do potencial de inversão que pode ser visto directamente nos traços de fotocorrente adquiridos. A avaliação dos potenciais de reversão a partir de múltiplas medições quantifica a mudança de potencial de reversão dramática causada pela variação da concentração de cloreto externo ( Figura 2B ). Surpreendentemente, os potenciais de reversão medidos correspondem directamente a potenciais de Nernst calculados para cloreto ( Figura 2C ) confirmando a elevada selectividade de cloreto de Ps ACR1. T &quot;fo: keep-together.within-page =&quot; 1 &quot;&gt; <img alt="Figura 2" src="/files/ftp_upload/55497/55497fig2.jpg" /> Figura 2: Selectividade de cloreto de Ps ACR1 . ( A ) Traços actuais representativos de Ps ACR1 em células HEK293. A concentração de cloreto intracelular foi mantida a 120 mM, enquanto que a concentração extracelular variou de 150 mM (verde) a 10 mM (púrpura) cloreto por substituição parcial de NaCl com Na-aspartato. O potencial de retenção aumentou em passos de 20 mV de -80 mV para +40 mV (corrigido LJP). ( B ) Relações tensão-tensão médias (n = 6 correspondentes às condições em A). ( C ) Potenciais de reversão (as linhas centrais indicam medianas, os limites de caixa indicam os percentis 25 e 75, os bigodes estendem 2 vezes o desvio padrão e os círculos indicam pontos de dados únicos) extraídos das relações tensão-corrente. Para o cloreto de alta(Verde), o valor foi interpolado linearmente entre -20 mV e 0 mV de potencial de retenção, enquanto que o valor foi extrapolado linearmente acima de +40 mV (roxo, tracejado, painel B). <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55497/55497fig2large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins at JoVE.com

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

Full-cell Patch-clamp Gravações para determinação eletrofisiológica de Ion Selectivity em Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate ...

whole-cell-patch-clamp-recordings-for-electrophysiological

Nanyang Technological University

Research

JoVE Journal

Neuroscience

17.0K visualizações.  Humboldt-Universität zu Berlin. This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

Vídeo: Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

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  ...

Neurociência

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 ...

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.

Isolamento de neurônios sensoriais de<em&gt; Aplysia californica</em&gt; Para Remendo Recordings Grampo de Correntes 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.

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 ...

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.

Whole-cell Recordings patch-clamp de Morphologically- e identificados Neuroquimicamente hipocampo Interneurônios

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

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

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

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

Emparelhados Recordings células inteiras em organotï¿½icas fatias do hipocampo

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

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

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

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

Eletrofisiológica e Caracterização Morfológica de Neuronal Microcircuits em aguda fatias do cérebro do Usando Emparelhados patch-clamp Recordings

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

Whole-cell Patch-clamp Recordings in Brain Slices

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

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

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 ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

Método eletrofisiológico para gravações de tensão de célula inteira de<em&gt; Drosophila</em&gt; Fotorreceptores

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

This protocol describes methods to record the auditory brainstem response from postnatal rat pups. To examine the functional development of outer hair cells, the experimental procedure of whole-cell patch clamp recording in isolated outer hair cells is described step-by-step.

Auditivo de tronco encefálico e exterior cabelo célula célula inteira Patch Clamp gravação em ratos pós-natal

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...