Financiado pelo NIH (NS-18205).

1. Preparação de peixes para Recordings emparelhados <ol><li> Antes de iniciar o procedimento, preparar cinco soluções-chave, que incluem: Solução de Bath, Bath Solution com tricaina (MS222), Solução Banheira com Formamida, Solução Neuron Interno e Solução Muscle Interna. </li><li> Em seguida, recolher uma única zebrafish larval entre as idades de 72 a 96 horas e coloque em banho com solução tricaina. Dentro de um minuto de exposição ao anestésico, os peixes não irá responder ao toque. </li><li> Coloque o peixe em cima de um prato de plástico e, em um estereomicroscópio para decapitar com uma lâmina de duplo fio de navalha. </li><li> Transfira o peixe para uma câmara de gravação de vidro revestidas com Sylgard e preenchido com solução de Bath. Aperte em cada extremidade, inserindo os pinos de tungstênio eletroliticamente afiada através da notocorda. </li><li> Criar um retalho de pele perto da extremidade rostral dos peixes com um pino que irá proporcionar uma aderência adequada para um par de fórceps bem. Retire a pele sobrejacente segurando-o perto do pino rostral com um par de fórceps bem. Descasque lentamente na direção caudal para remover a pele. </li><li> Tratar os peixes com 3 ml de solução Banheira com Formamida por 3-5 minutos para bloquear as contrações musculares que possam interferir com as gravações. Lavar 5 vezes com Solução de Bath normal. Remover parte da camada superficial do músculo mais de 5-6 segmentos suavemente a demolição com o lado de um pino de tungstênio. </li><li> Transfira o peixe para um microscópio vertical localizado em uma gaiola de Faraday para proteger ruído elétrico. O microscópio é equipado com um palco fixo, longa distância de trabalho objetiva de 40x e zoom de 4x para 0,5. O microscópio é montado em um palco motorizado tradução XY, a fim de mover a preparação entre nervo e músculo sob alta potência. O uso de contraste de interferência diferencial óptica ajuda a visualizar os neurônios. </li><li> Abaixo de 0,5 x de zoom, use um furo de largura de 15 micron pipeta para remover muscular sobrejacente e expor a medula espinhal. Pressão negativa é aplicada através de uma seringa descartável e cc 3 células musculares são removidos um a um. Este procedimento é repetido para 5-6 segmentos dorsal do músculo esquelético. O mesmo largo furo pipeta é usado também para remover a parte superior duas camadas de músculo ventral para obter músculo alvo rápida do esqueleto. </li><li> Isso completa a preparação para as gravações emparelhado eo zoom microscópio é aumentada para cerca de 1.6x. </li></ol> 2. Gravações pareadas de CaP Motor-neurônio e células musculares Alvo <ol><li> Os segmentos de limpeza são digitalizados para localizar os neurônios CaP. Cada hemi-segmento da medula espinhal contém uma grande diâmetro micron 10, CaP motor-neurônio. Esta soma teardrop forma está localizado superficialmente sob a dura-máter espinhal perto do final mais caudal do limite segmentar. Um neurônio CaP é escolhido para a gravação e do músculo alvo ventral no segmento adjacente caudal é verificado para verificar a integridade. </li><li> Dois eletrodos patch são posicionados para a gravação, um para o neurônio e um segundo para o músculo. O eletrodo superior tem uma vela rasa para a penetração da dura espinhal dura e abertura de ponta de cerca de 2 microns. Quanto menor eletrodo tem uma íngreme taper de baixa resistência de gravação braçadeira de tensão do músculo esquelético </li><li> Posição do eletrodo de neurônio em um ângulo de aproximadamente 30 graus para penetrar na dura espinhal, cerca da metade do segmento rostral à neurônio CaP selecionado. Avançar o eletrodo usando um manipulador disco axial ao aplicar uma pressão contínua suave positivo de aproximadamente 60 milímetros de mercúrio. Como eletrodo rompe a dura-máter, a PAC neurônio pode ser deslocada pela perfusão do eletrodo, exigindo um reajuste de foco. </li><li> Quando o eletrodo toca o CaP neurônio pressão soma positiva é liberado e, geralmente, resulta na formação imediata de um selo gigaohm. Porém, em alguns casos em que um selo não se forma, torna-se necessário aplicar uma leve pressão negativa. Após a formação selo, o potencial de eletrodo é ajustada para 80mV-e aplicação de pressão negativa rupturas da membrana celular. O neurônio CaP é caracterizada por uma resistência de entrada de 140-180 Mega-ohms e um potencial de ação que ultrapassa 40 milivolts. Este é testada injetando gradativamente aumentando passos despolarizantes em pinça de corrente até que o potencial de ação é eliciada. </li><li> Em seguida, o microscópio é transferida para o músculo ventral usando o tradutor XY motorizada, sob alta potência. A célula muscular rápido é escolhido a partir do campo de destino. </li><li> O eletrodo é avançado na direção do músculo na célula muscular alvo sob pressão positiva, que quando liberado, forma um selo gigaohm. O potencial de eletrodo é ajustado a -50 milivolts para inativar os canais de sódio e sob sugar suave a membrana da célula é rompido. A queda na resistência à 100-200 Mega-ohms eo súbito aparecimento dos transientes de entrada capacitiva grandes sinais. </li><li> Para testarpara a gravação emparelhado, o motor-neurônio é despolarizada por injeções de forma incremental maior de corrente até um potencial de ação é eliciada. Neste ponto, uma placa terminal atual deve ser atingida em músculo. Estimulação continuada do motorneuron a 1 Hertz resultados em correntes de placa terminal sem falhas. Como a frequência de estímulo é aumentada para 100 Hertz as correntes placa terminal flutuar em amplitude e passar pelo esgotamento funcional dentro de 10 segundos de estimulação. </li></ol> 3. Resultados representante Normalmente, o neurônio é a gravação estável por até uma hora, mas as células musculares se deteriorar conforme refletido como um aumento na resistência série. Quando isso ocorre o experimento é terminado ou outra célula muscular é remendo preso. Todas as gravações devem ser concluídas dentro de uma hora. <img src="/files/ftp_upload/2351/2351fig1.jpg" alt="Figura 1" /> Figura 1. Recordings do potencial de ação motorneuron (AP, em cima) e placa terminal muscular associada atual (EPC, baixo). O potencial de ação de um neurônio PAC deve ultrapassar 40 mV eo EPC deve subir a 300 ms e decadência ao longo de um curso de tempo exponencial com uma constante de tempo inferior a 1 ms. <table border="1"><tbody><tr><td> Nome </td><td> Receita </td></tr><tr><td> Solução de banho (em mM) </td><td> 134 NaCl, 2,9 KCl, CaCl 2 2,1, 1,2 MgCl 2, 10 Glucose, 10 Na-HEPES, pH 7.8 </td></tr><tr><td> Solução de banho com tricaina </td><td> Solução banho contendo tricaina 0,02% </td></tr><tr><td> Solução de banho com Formamida </td><td> Solução banho contendo formamida 2M </td></tr><tr><td> Neurônio solução interna (em mM) </td><td> 115 K-gluconato, 15 de KCl, MgCl 2 2, 10 K-HEPES, 5 K-EGTA, 4 Mg-ATP, pH 7,2 </td></tr><tr><td> Solução muscular interna (em mM) </td><td> 120 KCl, 10 K-HEPES, 5 BAPTA, pH 7,4 </td></tr></tbody></table> Tabela 1. Solutions.

<ol>
	<li>Fetcho, J. R., O'Malley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+networks+in+behaving+animals.">Imaging neuronal networks in behaving animals.</a> Current Opinion in Neurobiology. 7, 832-838 (1997).</li><li>Fetcho, J. R., S, H. i. g. a. s. h. i. j. i. m. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+and+genetic+approaches+toward+understanding+neuronal+circuits+in+zebrafish.">Optical and genetic approaches toward understanding neuronal circuits in zebrafish.</a> Integr Comp Biol. 44, 57-70 (2004).</li><li>Iba&#241;ez-Tallon, I., Wen, H., Miwa, J., Xing, J., Aslantas-Tekinay, A., Ono, F., Brehm, P., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tethering+naturally+occurring+peptide+toxins+for+cell+autonomous+modulation+of+ion+channels+and+receptors+in+vivo.">Tethering naturally occurring peptide toxins for cell autonomous modulation of ion channels and receptors in vivo.</a> Neuron. 43, 305-311 (2004).</li><li>Li, W., Ono, F., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+measurements+of+presynaptic+release+in+mutant+zebrafish+lacking+postsynaptic+receptors.">Optical measurements of presynaptic release in mutant zebrafish lacking postsynaptic receptors.</a> J. Neuroscience. , 23-10467 (2003).</li><li>Ono, F., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholine+receptors+direct+rapsyn+clusters+to+the+neuromuscular+synapse+in+zebrafish.">Acetylcholine receptors direct rapsyn clusters to the neuromuscular synapse in zebrafish.</a> J. Neuroscience. 24, 475-5481 (2004).</li><li>Ono, F., Shcherbatko, A., Higashijima, S., Fetcho, J., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paralytic+zebrafish+lacking+ACh+receptors+fail+to+localize+rapsyn+clusters+to+the+synapse.">Paralytic zebrafish lacking ACh receptors fail to localize rapsyn clusters to the synapse.</a> J. Neuroscience. 21, 5439-5448 (2001).</li><li>Ono, F., Shcherbatko, A., Higashijima, S., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+motility+mutant+twitch+once+reveals+new+roles+for+rapsyn+in+synaptic+function.">The zebrafish motility mutant twitch once reveals new roles for rapsyn in synaptic function.</a> J. Neuroscience. 22, 6491-6498 (2002).</li><li>Wang, M., Wen, H., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+neuromuscular+synapses+in+the+zebrafish+choline-acetyltransferase+mutant+bajan.">Function of neuromuscular synapses in the zebrafish choline-acetyltransferase mutant bajan.</a> J Neurophysiol. , 100-104 (2008).</li><li>Wen, H., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paired+motor-neuron+muscle+recordings+in+zebrafish+test+the+receptor+blockade+model+for+shaping+synaptic+current.">Paired motor-neuron muscle recordings in zebrafish test the receptor blockade model for shaping synaptic current.</a> J. Neuroscience. 25, 8104-8111 (2005).</li></ol>

Não há conflitos de interesse declarados.

Temos vindo a utilizar zebrafish gravações emparelhados (Wen e Brehm, 2005), principalmente para estudar os processos de liberação de vesículas e reciclagem durante a estimulação de alta freqüência. Este processo é interrompido em mutantes motilidade em que a transmissão sináptica normal é comprometida devido a mutações pontuais nos principais componentes sináptica. Por exemplo, mutações que perturbam agregados receptor pós-sináptico (Ono et al., 2001, 2002, 2004) e de síntese do transmissor pré-sináptico (Wang et al., 2008) resultar em não-coordenada de natação. Gravações emparelhado fornecer as informações que possam identificar a origem do defeito funcional, que liga comportamento, genética e fisiologia. Agora deve ser possível dissecar ainda mais os processos subjacentes a transmissão sináptica por meio da expressão transitória do CaP motor neurônios utilizando promotores criteriosa. Desta forma dominante genes mutados negativa ou pode ser especificamente expressas nestes neurônios e suas conseqüências comportamentais e eletrofisiológicos determinado. A vantagem adicional oferecida pela configuração da célula permite que toda a diálise da soma CaP com agentes que podem potencialmente alterar a reciclagem das vesículas, como toxinas clostridial e buffers de cálcio. Devido à curta distância entre o soma eo músculo, a difusão deve ocorrer bem dentro do prazo de gravações. O potencial desta preparação só começou a ser aproveitado. A transparência dos peixes neste local idade e superficial de sinapses também facilita a utilização de instrumentos ópticos (Fetcho e O&#39;Malley, 1997; Fetcho e Higashijima, 2004). Temos sido muito bem sucedida na medição de endo e exocitose nos peixes que vivem usando corantes FM e indicadores genéticos, tais como Synaptophluorin (Li et al., 2003). Além disso, as toxinas conjugado fluorescente pode ser usado para rotular proteínas sinápticas para geração de imagens de peixes vivos (Ibanez-Tallon et al., 2004). Dada a simplicidade desta preparação, é uma grande promessa para futuros estudos.

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<td>Pneumatic transducer tester</td>
<td>&nbsp;</td>
<td>Fluke Biomedical Instruments</td>
<td>DPM1B</td>
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<td>0.002 x 3 inch tungsten rod</td>
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<td>A-M Systems Inc</td>
<td>715000</td>
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<td>Sylgard 184 Elastomer</td>
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<td>Dow Corning Corp.</td>
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<td>The thickness of application will affect the DIC optics</td>
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paired patch clamp recordings from motor-neuron and target skeletal muscle in zebrafish

Larval zebrafish representar o sistema primeiro modelo vertebrados para permitir a gravação simultânea patch clamp um músculo do neurônio motor e alvo da coluna vertebral. Esta é uma conseqüência direta da acessibilidade para os dois tipos de células ea capacidade de distinguir visualmente o CaP única segmentar motor-neurônio com base na morfologia e localização. Este vídeo demonstra os métodos microscópicos utilizados para identificar uma CaP motor-neurônio e células musculares alvo, bem como as metodologias para a gravação de cada tipo de célula. A identificação do tipo CaP motor-neurônio é confirmado por um ou outro recheio corante ou pelas características biofísicas, como forma de onda do potencial de ação e resistência de entrada da célula. Motor-neurônio gravações rotineiramente durar uma hora permitindo gravações a longo prazo a partir de várias células musculares diferentes alvo. Controle sobre o padrão de disparo dos neurônios motor permite medições da freqüência de dependência da transmissão sináptica na junção neuromuscular. Devido a um tamanho grande quântica eo baixo nível de ruído fornecida pelo clamp toda célula de tensão, todos os eventos unitária podem ser resolvidos no músculo. Este recurso permite estudo de propriedades básicas sináptica, como propriedades de liberação, a reciclagem de vesícula, bem como a depressão sináptica e facilitação. As vantagens oferecidas por este in vivo preparação eclipse anterior modelo de sistemas neuromuscular onde estudou os neurônios motores são geralmente estimuladas por eletrodos extracelulares e os músculos são grandes demais para grampo toda célula patch. A preparação do peixe-zebra é passível de análise eletrofisiológica combinando com uma grande variedade de abordagens, incluindo linhagens transgênicas, knockdown morfolino, intervenção farmacológica e in vivo de imagens. Estas abordagens, juntamente com o crescente número de modelos de doenças neuromusculares fornecidos por linhas mutantes de peixe-zebra, abrir a porta para uma nova compreensão do ser humano doenças neuromusculares.

Watch this Scientific Journal Video about Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish at JoVE.com

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish representar o sistema primeiro modelo vertebrados para permitir a gravação simultânea patch clamp um músculo do neurônio motor e alvo espinhal esquelético. Este vídeo demonstra os métodos microscópicos utilizados para identificar uma CaP segmentar do neurônio motor e células do músculo-alvo, bem como as metodologias para a gravação de cada tipo de célula.

Gravações patch emparelhado Grampo da Motor-neurônio e músculo alvo ósseas em Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

paired-patch-clamp-recordings-from-motor-neuron-target-skeletal

Research

JoVE Journal

Neuroscience

17.0K visualizações.  Oregon Health and Sciences University. Larval zebrafish representar o sistema primeiro modelo vertebrados para permitir a gravação simultânea patch clamp um músculo do neurônio motor e alvo espinhal esquelético. Este vídeo demonstra os métodos microscópicos utilizados para identificar uma CaP segmentar do neurônio motor e células do músculo-alvo, bem como as metodologias para a gravação de cada tipo de célula.

Vídeo: Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

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

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 ...

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 ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

Subcelulares Gravações patch-clamp do domínio somatodendrítico da substância negra dopamina Neurônios

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...