Esta pesquisa foi apoiada por fundos de inicialização UT Southwestern (SK).

<ol>
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B., Messer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+composition+of+experimental+solutions+on+neuronal+survival+during+rat+brain+slicing.">Effect of composition of experimental solutions on neuronal survival during rat brain slicing.</a> Exp Neurol. 131 (1), 133-143 (1995).</li><li>Tanaka, Y., Tanaka, Y., Furuta, T., Yanagawa, Y., Kaneko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+cutting+solutions+on+the+viability+of+GABAergic+interneurons+in+cerebral+cortical+slices+of+adult+mice.">The effects of cutting solutions on the viability of GABAergic interneurons in cerebral cortical slices of adult mice.</a> J Neurosci Methods. 171 (1), 118-125 (2008).</li><li>Ye, J. H., Zhang, J., Xiao, C., Kong, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch-clamp+studies+in+the+CNS+illustrate+a+simple+new+method+for+obtaining+viable+neurons+in+rat+brain+slices:+glycerol+replacement+of+NaCl+protects+CNS+neurons.">Patch-clamp studies in the CNS illustrate a simple new method for obtaining viable neurons in rat brain slices: glycerol replacement of NaCl protects CNS neurons.</a> J Neurosci Methods. 158 (2), 251-259 (2006).</li><li>Kourrich, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+interaction+between+sigma-1+receptor+and+Kv1.2+shapes+neuronal+and+behavioral+responses+to+cocaine.">Dynamic interaction between sigma-1 receptor and Kv1.2 shapes neuronal and behavioral responses to cocaine.</a> Cell. 152 (1-2), 236-247 (2013).</li><li>Kourrich, S., Klug, J. R., Mayford, M., Thomas, M. 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Nenhum dos autores tem interesses concorrentes ou interesses conflitantes.

Este protocolo descreve o procedimento básico para a realização de experiências de célula inteira de patch-clamp em neurónios em fatias de cérebro. No entanto, a complexidade, o potencial e sensibilidade desta técnica não pode ser totalmente descrito neste artigo. Aqui, tentamos delinear os passos mais básicos e sublinhado parâmetros importantes que devem ser controlados para alcançar gravações de células inteiras de sucesso e rigorosos. Para mais aprendizado teórico, muitos livros e artigos foram public...

A técnica de patch-clamp, uma técnica eletrofisiológica que tem sido desenvolvido no final de 1970 1,2, é a principal ferramenta para o estudo de funções de canal de iões simples ou múltiplas no tecido vivo. Entre as diferentes configurações de patch que podem ser obtidos, gravações de célula inteira de patch-clamp permitir o estudo do comportamento eléctrico de uma parte substancial do neurónio. Classicamente, esta técnica é realizada in vitro, quer em fatias de cérebro, os neurónios dissociados de fresco, quer em modelos de cultura de células 3. Quando realizada em neurônios em fatias de cérebro, esta técnica apresenta várias vantagens. Em particular: (i) os neurónios são registados nos circuitos do cérebro relativamente conservadas que, em certa medida, e em comparação com as preparações de cultura de células, proporcionam um ambiente que é fisiologicamente relevante 3. Isto permite a captura precoce, ou mesmo de monitoramento em tempo real, eventos celulares e moleculares que são acionados por qualquer tipo de pharmacolog agudamanipulações iCal - uma resolução temporal que não pode ser conseguido usando clássica em condições in vivo; (ii) capacidade de identificar visualmente as regiões do cérebro em fatias de cérebro permite alta especificidade regional 3, tanto para a região do cérebro estudada e para os neurônios específicos quando eles expressam marcadores fluorescentes; (iii) o acesso ao espaço intracelular da célula através da abertura de uma parte significativa da membrana de plasma (em contraste com a perfuração da membrana com uma micropipeta afiado para gravações intracelulares) 4. Por sua vez, isto permite que o conteúdo ou concentração de iões específicos que constituem a solução interna de ser modificado de modo alvos moleculares ou mecanismos celulares pode ser estudada em condições diferentes. Por exemplo, ao estabelecer a configuração de célula inteira, qualquer agente farmacológico específico (por exemplo, antagonistas) que se pode adicionar ao micropipeta de gravação (remendo pipeta) solução difundirá directamente para o citoplasma e agir sobre a sua Putative alvos intracelulares, sem alterar a função alvo em células vizinhas. Além disso, em comparação com a gravação micropipeta afiada, a grande abertura na ponta do eléctrodo de patch clamp proporciona uma resistência mais baixa, menos ruído concorrentes, e, assim, um melhor acesso eléctrico para o interior da célula 4. No entanto, note que o da grande abertura na ponta da pipeta podem levar a célula de diálise, e, assim, a perda da maquinaria molecular intracelular que pode ser crítico para a expressão dos fenómenos biológicos que estão sob 5,6 estudo. Neste caso, as gravações de eléctrodos afiadas pode ser mais adequado. Este tipo de gravações requer micropipetas com uma poro que é muito menor do que os utilizados para as gravações de células inteiras, evitando deste modo a maior parte da permuta iónica entre o espaço intracelular e a solução da pipeta interna. Qualquer forma de experiência (aguda ou crónica), incluindo a aprendizagem 7-10, exposição a drogas de abuso 11,12, o stress 13,14, etc, pode alterar diversos aspectos da função neuronal em regiões específicas do cérebro. Porque estas alterações, muitas vezes exigem tempo para se desenvolver (horas ou dias), gravações de célula inteira em fatias de cérebro de animais que tenham sido submetidos a uma experiência específica permitir aos pesquisadores identificar essas mudanças. Basicamente, muitos (se não todos) os componentes que participam de funções neuronais (por exemplo, canais iónicos activados por ligandos, canais de iões dependentes da voltagem, transportadores de neurotransmissores), e assim a actividade circuito cerebral e comportamento, pode ser alterada por experiência (experiência-dependente plasticidade) 10,15-17. A nivel neuronal, a actividade circuito cerebral surge a partir de interacções constantes entre sináptica (por exemplo, glutamato) de transmissão e os factores de excitabilidade celular intrínseco (por exemplo, canais iónicos axosomato-dendrítica: de sódio, Na +, potássio, K +, e de cálcio, Ca 2+ ). Sob condições específicas, utilizando whole de células de patch-clamp técnicas electrofisiológicas, alterações de sinal específica das alterações na excitabilidade sináptica vs intrínseca pode ser isolado. Na maioria dos casos, a excitabilidade sináptica é avaliada utilizando a técnica de tensão-grampo de célula inteira. Este modo de gravação permite a medição de correntes de iões [por exemplo, mediados por receptores do ácido α-amino-3-hidroxi-5-metil-4-isoxazolpropiónico ( os receptores de AMPA) e receptores de N-metil-D-aspártico (NMDA)] receptores através da membrana plasmática neuronal, enquanto que prendem o potencial de membrana a uma voltagem conjunto. Aqui, os experimentadores usar soluções internas micropipeta que contêm césio (Cs +), um bloqueador ampla de canais de K + (principais fatores de excitabilidade intrínsecas). Após o estabelecimento da configuração de célula inteira, a difusão de Cs + no espaço intracelular irá bloquear os canais de K +, e, assim, vai permitir que ambos um espaço relativamente eficiente e pré-braçadeiradesabafar influência de fatores intrínsecos excitabilidade sobre outras medidas. Questões de espaço-clamp, ou seja, a dificuldade de tensão-clamp toda a célula, surgem quando a gravação de células irregular em forma (por exemplo, neurônios), e particularmente neurônios com um dendrítica vasto e complexo Arbor 18,19. Por causa da braçadeira de tensão somática controles mal tensão na árvore dendrítica de neurônios, vários aspectos de sinais elétricos dendríticas em estudo são distorcidos de uma forma dendrítica dependente da distância. Combinado com ferramentas farmacológicas tais como picrotoxina (ácido gama-aminobutírico, o GABA Um antagonista do receptor) ou ácido quinurï¿½ico (largo bloqueador de receptores de glutamato) dissolvido na solução extracelular (fluido cérebro-espinal artificial, ACSF), esta técnica permite a medição de glutamato receptor- e correntes de GABA A R-mediada, respectivamente. Em contraste, a excitabilidade intrínseca é normalmente avaliado em modo de gravação actual-clamp.Ao contrário de gravação voltagem-grampo, este modo de gravação permite a medição de variações no potencial de membrana induzidas por correntes de iões que fluem através da membrana plasmática neuronal. Normalmente, a alteração na excitabilidade intrínseca é avaliado através de alterações na capacidade de neurônios para gerar potenciais de ação, o que requer Na + e canais de K +. Portanto, quando se realiza gravações de corrente-clamp, micropipetas são preenchidos com uma solução interna que contém K + em vez de Cs +. Combinados com agentes farmacológicos que bloqueiam a glutamato e GABA A correntes mediadas pelo receptor dissolvidos em ACSF, este delineamento experimental permite a medição da contribuição de factores intrínsecos (por exemplo, canais de K +) para o disparo neuronal sem serem contaminados por potenciais alterações na excitabilidade sináptica factores. Este artigo irá descrever as etapas básicas necessárias processuais to (i) preparar fatias cerebrais saudáveis; (Ii) obter uma configuração de célula inteira, e (iii) monitorar os parâmetros básicos para avaliar sináptica e excitabilidade intrínseca.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Isolated pulse stimulus generator</td>
			<td style="width:183px;">A.M.P.I</td>
			<td style="width:139px;">Master-8</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isolation unit (ISO-Flex)</td>
			<td>A.M.P.I</td>
			<td>ISO-Flex</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer controlled Amplifier&#160;</td>
			<td>Molecular Devices</td>
			<td>Multiclamp 700B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital Acquisition system</td>
			<td>Molecular Devices</td>
			<td>Digidata 1500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td>Olympus</td>
			<td>BX-51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>MPC-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chamber and in-line Heater</td>
			<td>Warner Instruments</td>
			<td>TC-344B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome Slicer</td>
			<td>Leica&#160;</td>
			<td>VT1000 S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette Puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaging Camera</td>
			<td>Q Imaging</td>
			<td>QIClick-F-M-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Narishige pipette puller PC-10</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass capillaries</td>
			<td>WPI</td>
			<td>TW150F-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slice hold-down (harp)</td>
			<td>Warner Instruments</td>
			<td>64-0255</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slice Chamber</td>
			<td>Warner Instruments</td>
			<td>RC-26</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nonmetallic syringe needle</td>
			<td>World Precision Instruments</td>
			<td>MF28G67-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe filters</td>
			<td>Nalgene</td>
			<td>176-0045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glue Gun</td>
			<td style="width:183px;">Home Depot</td>
			<td style="width:139px;">various</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gas dispersion tube</td>
			<td>Ace Glass Inc.</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Decapitation scissors</td>
			<td>Home Depot</td>
			<td>100649198</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel Handle #3</td>
			<td>World Precision Instruments</td>
			<td>500236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small straight sharp tips scissors</td>
			<td>World Precision Instruments</td>
			<td>14218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vessel canulation forceps&#160;</td>
			<td>World Precision Instruments</td>
			<td>500453</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved hemostatic forceps</td>
			<td>World Precision Instruments</td>
			<td>501288</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Economy Tweezers #3</td>
			<td>World Precision Instruments</td>
			<td>501976-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spatula</td>
			<td>Fisher Scientific</td>
			<td>14357Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scooping spatula</td>
			<td>Fisher Scientific</td>
			<td>14-357Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dish</td>
			<td>Fisher Scientific</td>
			<td>08-747B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter paper</td>
			<td>Lab Depot</td>
			<td>CFP1-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Name of Material/ Equipment</td>
			<td style="width:183px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;"></td>
			<td style="width:183px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;">Cs-Gluconate internal solution (pH 7.2&#8211;7.3, 280&#8211;290 mOsm)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-gluconic acid 50%&#160;</td>
			<td>Sigma Aldrich/various</td>
			<td>G1951</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cesium-OH (CsOH) 50%&#160;</td>
			<td>Sigma Aldrich/various</td>
			<td>232041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl, 2.8 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES, 20 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA, 0.4 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tetraethylammonium-Cl, 5 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>T2265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2GTP, 0.3 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgATP, 2 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Name of Material/ Equipment</td>
			<td style="width:183px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;">K-Gluconate internal solution (pH 7.2&#8211;7.3, 280&#8211;290 mOsm)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">K D-gluconate, 120 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>G4500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl, 20 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES, 10 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA, 0.2 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2</td>
			<td>Sigma Aldrich/various</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2GTP, 0.3 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgATP, 2 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Name of Material/ Equipment</td>
			<td style="width:183px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;">Standard artificial cerebrospinal fluid (ACSF, osmolarity &#8776; 300-310 mOsm)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl, 2.5 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl, 119 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaH2PO4-H20, 1 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaHCO3, 26.2 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>S8875</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose, 11 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4-7H2O, 1.3 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>230391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2-2H20, 2.5 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>C3881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Name of Material/ Equipment</td>
			<td style="width:183px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Additional compounds used for solutions preparation</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KOH</td>
			<td>various</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kynurenic acid</td>
			<td>Sigma Aldrich/various</td>
			<td style="width:139px;">K3375</td>
			<td></td>
		</tr>
	</tbody>
</table>

whole-cell patch-clamp recordings in brain slices

Whole-cell patch-clamp de gravação é uma técnica electrofisiológico que permite o estudo das propriedades eléctricas de uma parte substancial do neurónio. Nesta configuração, a micropipeta está em contacto apertado com a membrana celular, o que evita a fuga de corrente e, assim, fornece medições mais precisas corrente iónica do que o método de gravação eléctrodo afiado intracelular previamente utilizado. Classicamente, a gravação de células inteiras pode ser realizada sobre os neurónios em vários tipos de preparações, incluindo modelos de cultura celular, os neurónios dissociados, neurónios em fatias de cérebro e em animais anestesiados intactos ou acordado. Em resumo, esta técnica tem imensamente contribuído para a compreensão das propriedades biofísicas passivas e activas das células excitáveis. Uma importante vantagem desta técnica é que ele proporciona informação sobre a forma específica manipulações (por exemplo, farmacológico, plasticidade induzida pelo experimentador) podem alterar ou C funções neuronais específicashannels em tempo real. Além disso, a abertura significativa da membrana plasmática permite que a solução da pipeta interna para difundir livremente no citoplasma, proporcionando meios para a introdução de medicamentos, por exemplo, agonistas ou antagonistas de proteínas intracelulares específicos, e manipular esses alvos, sem alterar as suas funções em células vizinhas. Este artigo incidirá sobre gravação de célula inteira executada em neurónios em fatias de cérebro, uma preparação que tem a vantagem de gravação de neurónios no cérebro circuitos relativamente bem conservados, isto é, num contexto fisiologicamente relevante. Em particular, quando combinado com farmacologia disso, esta técnica é uma ferramenta poderosa que permite a identificação de neuroadaptações específicas que ocorreram na sequência de qualquer tipo de experiências, como a aprendizagem, a exposição a drogas de abuso, e stress. Em resumo, as gravações de células inteiras de patch-clamp em fatias de cérebro fornecer meios para medir em ex vivo de preparação de mudanças de longa duraçãoem funções neuronais que se desenvolveram em animais acordados intactas.

Temperatura, um fator que é facilmente controlada pelo experimentador, influencia as propriedades biofísicas de canais iônicos e receptores, e, assim, a forma de onda de correntes pós-sinápticas (PSCs) (EPSC e iPSCs) e a capacidade dos neurônios para provocam picos. Figura 3 e a Figura 4 mostram o efeito da temperatura sobre o disparo neuronal e a inclinação da EPSCS evocados (eEPSCs), respectivamente. O padrão de disparo (Figura 3)</str...

Watch this Scientific Journal Video about Whole-cell Patch-clamp Recordings in Brain Slices at JoVE.com

Whole-cell Patch-clamp Recordings in Brain Slices

Este protocolo descreve passos processuais básicas para a realização de célula inteira gravações de patch-clamp. Esta técnica permite o estudo do comportamento eléctrico de neurónios, e quando realizados em fatias do cérebro, permite a avaliação de várias funções neuronais a partir de neurónios que ainda estão integrados nos circuitos do cérebro relativamente bem conservadas.

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 ...

whole-cell-patch-clamp-recordings-in-brain-slices

Research

JoVE Journal

Neuroscience

59.3K visualizações.  University of Texas Southwestern Medical Center.  Este protocolo descreve passos processuais básicas para a realização de célula inteira gravações de patch-clamp. Esta técnica permite o estudo do comportamento eléctrico de neurónios, e quando realizados em fatias do cérebro, permite a avaliação de várias funções neuronais a partir de neurónios que ainda estão integrados nos circuitos do cérebro relativamente bem conservadas. 

Vídeo: Whole-cell Patch-clamp Recordings in Brain Slices

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 ...

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 ...

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

A Multi-eletrodo Sistema patch-clamp assistida por computador

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

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 for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

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 ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

Aplicação de correia de patch automatizada com guia de imagem para estudo de neurônios em fatias de cérebro

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

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 ...