Name: external excitation of neurons using electric and magnetic fields in one- and two-dimensional cultures
Uploaded: 2017-05-07T14:00:00
Description: Watch this Scientific Journal Video about External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures at JoVE.com

Os autores agradecem Ofer Feinerman, Fred Wolf, Menahem Segal, Andreas Neef e Eitan Reuveny para discussões muito úteis. Os autores agradecem Ilan Breskin e Jordi Soriano para o desenvolvimento de versões iniciais da tecnologia. Os autores agradecem a Tsvi Tlusty e Jean-Pierre Eckmann pela ajuda com os conceitos teóricos. Esta pesquisa foi apoiada pela Fundação Minerva, o Ministério da Ciência e Tecnologia, Israel, e pela Fundação Ciência de Israel subvenção 1320/09 e da Fundação Bi-Ciência Fundação 2008331.

Declaração de Ética: Os procedimentos envolvendo manuseio de animais foram feitos de acordo com as diretrizes do Comitê Institucional de Cuidados e Uso de Animais (IACUC) do Instituto Weizmann de Ciência e a lei israelense apropriada. O Instituto Weizmann é credenciado pela Associação de Avaliação e Acreditação do Laboratório Animal Care International (AAALAC). O Comitê Institucional de Cuidados e Uso Animal de Weizmann aprovou este estudo, conduzido com os neurônios do hipocampo. <p class="jove_title...

<ol>
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Padrão 1D é uma ferramenta importante que pode ser usado para uma variedade de aplicações. Por exemplo, utilizamos padrões 1D para criar portas lógicas a partir de culturas neuronais 29 e mais recentemente para medir o Chronaxie e Rheobase de neurônios do hipocampo de ratos 5 e a desaceleração da velocidade de propagação de sinal da atividade de disparo em neurônios do hipocampo de síndrome de Down em comparação com a Neurônios do hipocampo do tipo selvagem...

A interacção entre os neurónios e os campos eléctricos externos tem implicações fundamentais, bem como os práticos. Embora seja conhecido desde os tempos de Volta que um campo eléctrico aplicado externamente pode excitar tecido, os mecanismos responsáveis para a produção de um potencial de acção resultante em neurónios são apenas recentemente começando a ser desvendado 1, 2, 3, 4. Isto inclui encontrar respostas a perguntas sobre o mecanismo que causa despolarização do potencial de membrana, o papel das propriedades da membrana e de canais iônicos, e até mesmo a região do neurônio que reage ao campo elétrico 2, 5. Terapêutico neuro 6, 7, 8, 9, <sup10 metodologias são particularmente dependentes desta informação, que pode ser crucial para a segmentação das áreas afectadas e para a compreensão dos resultados da terapia. Essa compreensão também pode ajudar no desenvolvimento de protocolos de tratamento e novas abordagens para estimulação de diferentes áreas do cérebro. A medição da interação dentro do cérebro in vivo acrescenta um componente importante a esta compreensão, mas é dificultada pela imprecisão e baixa controlabilidade das medições dentro do crânio. Em contraste, as medições em culturas podem ser facilmente realizadas em alto volume com alta precisão, excelente desempenho sinal-ruído e um alto grau de reprodutibilidade e de controle. Usando culturas uma grande variedade de propriedades neuronais do comportamento coletivo da rede pode ser elucidada 11 , 12 , 13 , 14 , </sup&gt; 15, 16. De igual modo, este sistema é bem controlada esperado ser altamente eficientes para elucidar o mecanismo pelo qual outros métodos de estimulação trabalhar, por exemplo, como a abertura do canal durante a estimulação óptica em neurónios optogenetically activas 17, 18, 19, é responsável pela criação de potencial de acção. Aqui, o foco está em descrever o desenvolvimento e compreensão das ferramentas que podem eficientemente excitam o neurônio através de um campo elétrico externo. Neste documento descreve-se a preparação de bidimensional e unidimensional culturas de hipocampo estampados, estimulação utilizando diferentes configurações e orientação de um campo eléctrico aplicado directamente por eléctrodos de banho, e, finalmente, a estimulação da bidimensional e modelado culturas unidimensionais por um tempo variando no campo magnético, o que induz um campo eléctrico5, 20, 21.

<table>
	<tbody>
		<tr>
			<td>APV</td>
			<td>Sigma-Aldrich</td>
			<td>A8054</td>
			<td>Disconnect the network. Mentioned in Section 2.4.2</td>
		</tr>
		<tr>
			<td>B27 supp</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>Plating medium. Mentioned in Section 1.1.1</td>
		</tr>
		<tr>
			<td>bicuculline</td>
			<td>Sigma-Aldrich</td>
			<td>14343</td>
			<td>Disconnect the network&#160;. Mentioned in Section 2.4.2</td>
		</tr>
		<tr>
			<td>Borax (sodium tetraborate decahydrate)</td>
			<td>Sigma-Aldrich</td>
			<td>S9640</td>
			<td>Borate buffer. Mentioned in Section 1.1.2</td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Frutarom LTD</td>
			<td>5550710</td>
			<td>Borate buffer. Mentioned in Section 1.1.2</td>
		</tr>
		<tr>
			<td>CaCl2 , 1M</td>
			<td>Fluka&#160;</td>
			<td>21098</td>
			<td>Extracellular recording solution&#160;. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Sigma-Aldrich</td>
			<td>C239</td>
			<td>Disconnect the network&#160;. Mentioned in Section 2.4.2</td>
		</tr>
		<tr>
			<td>COMSOL</td>
			<td>COMSOL Inc</td>
			<td>Multiphysics 3.5</td>
			<td>Numerical simulation. Mentioned in Section 3.5.2</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose, 1M</td>
			<td>Sigma-Aldrich</td>
			<td>65146</td>
			<td>Plating medium, Extracellular recording solution&#160;. Mentioned in Section 1.1.1&#160;&#160;&#160; 1.5.2</td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td>Cell Cultures. Mentioned in Section 1.2.4&#160;&#160;&#160; 1.2.6</td>
		</tr>
		<tr>
			<td>FCS(FBS)</td>
			<td>Gibco</td>
			<td>12657-029</td>
			<td>Plating medium. Mentioned in Section 1.1.1</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma-Aldrich</td>
			<td>F1141</td>
			<td>Bio Coating. Mentioned in Section 1.2.6</td>
		</tr>
		<tr>
			<td>Fluo4, AM</td>
			<td>Life technologies</td>
			<td>F14201</td>
			<td>Imaging of spontaneous or evoked activity&#160;. Mentioned in Section 1.5.1&#160;&#160;&#160; 1.5.3&#160;&#160;&#160; 1.5.5</td>
		</tr>
		<tr>
			<td>FUDR</td>
			<td>Sigma-Aldrich</td>
			<td>F0503</td>
			<td>Changing medium. Mentioned in Section 1.4.1</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Sigma-Aldrich</td>
			<td>G1272</td>
			<td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1</td>
		</tr>
		<tr>
			<td>GlutaMAX 100X</td>
			<td>Gibco</td>
			<td>35050-038</td>
			<td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1</td>
		</tr>
		<tr>
			<td>Hepes, 1M</td>
			<td>Sigma-Aldrich</td>
			<td>H0887</td>
			<td>Extracellular recording solution&#160;. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>HI HS&#160;</td>
			<td>BI</td>
			<td>04-124-1A</td>
			<td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1&#160;&#160;&#160; 1.4.1&#160;&#160;&#160; 1.4.2</td>
		</tr>
		<tr>
			<td>KCl,&#160; 3M</td>
			<td>Merck</td>
			<td>1049361000</td>
			<td>Extracellular recording solution. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>Laminin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td>Bio Coating. Mentioned in Section 1.2.6</td>
		</tr>
		<tr>
			<td>MEM x 1</td>
			<td>Gibco</td>
			<td>21090-022</td>
			<td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.4.1&#160;&#160;&#160; 1.4.2</td>
		</tr>
		<tr>
			<td>MgCl2 , 1M</td>
			<td>Sigma-Aldrich</td>
			<td>M1028</td>
			<td>Extracellular recording solution. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>NaCl, 4M</td>
			<td>Bio-Lab</td>
			<td>19030591</td>
			<td>Extracellular recording solution&#160;. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>Octadecanethiol</td>
			<td>Sigma-Aldrich</td>
			<td>01858</td>
			<td>Cleaning Cr-Au coated coverslips (1D cultures). Mentioned in Section 1.2.3</td>
		</tr>
		<tr>
			<td>Pluracare F108 NF Prill</td>
			<td>BASF Corparation&#160;</td>
			<td>50475278</td>
			<td>Bio-Rejection Coating, Bio Coating. Mentioned in Section 1.2.4&#160;&#160;&#160; 1.2.6</td>
		</tr>
		<tr>
			<td>Poly-L-lysine 0.01% solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;P47075</td>
			<td>Promote cell division. Mentioned in Section 1.1.4</td>
		</tr>
		<tr>
			<td>Sucrose, 1M</td>
			<td>Sigma-Aldrich</td>
			<td>S1888</td>
			<td>Extracellular recording solution&#160;. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>Thiol&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>1858</td>
			<td>Bio-Rejection Coating. Mentioned in Section 1.2.3</td>
		</tr>
		<tr>
			<td>URIDINE</td>
			<td>Sigma-Aldrich</td>
			<td>U3750</td>
			<td>Changing medium. Mentioned in Section 1.4.1</td>
		</tr>
		<tr>
			<td>Sputtering machine</td>
			<td>AJA International, Inc</td>
			<td>ATC Orion-5Series&#160;</td>
			<td>coating glass with thin layers of metal. Mentioned in Section 1.2.2</td>
		</tr>
		<tr>
			<td>Pen plotter&#160;</td>
			<td>Hewlett Packard&#160;</td>
			<td>HP 7475A</td>
			<td>Etching of pattern to the coated coverslip. Mentioned in Section 1.2.5</td>
		</tr>
		<tr>
			<td>Electrodes wires&#160;</td>
			<td>A-M Systems, Carlsborg WA</td>
			<td>767000</td>
			<td>Electric stimulation of neuronal cultures. Mentioned in Section 2.1&#160;&#160;&#160; 2.2&#160;&#160;&#160; 2.3&#160;&#160; 2.4.5</td>
		</tr>
		<tr>
			<td>Signal generator</td>
			<td>BKPrecision</td>
			<td>4079</td>
			<td>Shaping of the electric signal. Mentioned in Section 2.3</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Homemade</td>
			<td></td>
			<td>Voltage amplification of the signal from the signal generator to the electrodes. Mentioned in Section 2.3</td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>Matrix&#160;</td>
			<td>MPS-3005 LK-3&#160;</td>
			<td>Power supply to the sputtering machine. Mentioned in Section 1.2.2.3</td>
		</tr>
		<tr>
			<td>Transcranial magnetic stimulation</td>
			<td>Magstim, Spring Gardens, UK</td>
			<td>Rapid 2</td>
			<td>Magnetic stimulation of neuronal culture. Mentioned in Section 3.1&#160;&#160; 3.3&#160;&#160; 3.4</td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Cognis</td>
			<td>Versamid 140</td>
			<td>Casting of homemade coils. Mentioned in Section 3.4</td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Shell</td>
			<td>EPON 815&#160;</td>
			<td>Casting of homemade coils. Mentioned in Section 3.4</td>
		</tr>
		<tr>
			<td>Platinum wires 0.005&#39;&#39; thick; A-M Systems,&#160;</td>
			<td>&#160;Carlsborg WA</td>
			<td>&#160;767000</td>
			<td>Electric stimulation of neuronal cultures. Mentioned in Section 2.1</td>
		</tr>
		<tr>
			<td>Circular magnetic coil</td>
			<td>Homemade</td>
			<td></td>
			<td>Magnetic stimulation of neuronal culture. Mentioned in Section 3.3</td>
		</tr>
		<tr>
			<td>WaveXpress SW</td>
			<td>B&#38;K Precision&#160;</td>
			<td></td>
			<td>Waveform editing software. Mentioned in Section 2.1.32</td>
		</tr>
		<tr>
			<td>Xion Ultra 897</td>
			<td>Andor</td>
			<td></td>
			<td>Sensitive EMCCD camera. Mentioned in Section 2.4.4</td>
		</tr>
	</tbody>
</table>

external excitation of neurons using electric and magnetic fields in one- and two-dimensional cultures

Um neurónio irá disparar um potencial de acção, quando o seu potencial de membrana excede um determinado limiar. A actividade típica do cérebro, isto ocorre como resultado de entradas químicos às suas sinapses. No entanto, os neurônios também pode ser excitado por um campo eléctrico imposto. Em particular, aplicações clínicas recentes ativar os neurônios, criando um campo elétrico externo. Por isso, é de interesse para investigar como o neurônio responde ao campo externo eo que faz com que o potencial de ação. Felizmente, a aplicação precisa e controlada de um campo eléctrico externo é possível que as células neuronais embrionárias que são excisados, dissociadas e cultivadas em culturas. Isso permite que a investigação dessas questões em um sistema altamente reprodutível. Neste artigo algumas das técnicas utilizadas para aplicação controlada de campo elétrico externo em culturas neuronais são revistos. As redes podem ser unidimensional, ou seja padronizado em lineaformas R ou deixadas a crescer em todo o plano do substrato, e, assim, bidimensional. Além disso, a excitao pode ser criada por aplicação directa do campo eléctrico através de eléctrodos imersos no fluido (eléctrodos de banho) ou através da indução do campo eléctrico utilizando o criação remota de impulsos magnéticos.

<html> O protocolo apresentado permite fácil padronização das culturas neuronais. Quando é combinado com vários métodos desenvolvidos para a estimulação, que permite fazer medições de alguns intrínsecas propriedades neuronais como chronaxie e reobase 5, para comparar as propriedades dos neurónios saudáveis e doentes 27, para encontrar formas ideais para estimular culturas como uma função de sua estrutura e muitos mais novas ab...

Watch this Scientific Journal Video about External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures at JoVE.com

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

culturas neuronais são um bom modelo para o estudo de técnicas emergentes de estimulação do cérebro através de seus efeitos sobre os neurônios individuais ou uma população de neurônios. Aqui apresentados são diferentes métodos para a estimulação de culturas neuronais modeladas por um campo eléctrico produzido directamente por eléctrodos de banho ou induzida por um campo magnético variável no tempo.

Excitação externa de Neurônios Usando Campos Elétricos e Magnéticos em One e culturas bidimensionais

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

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Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Dissecção e Cultura de Neurônios da Medula Espinhal comissural embrionárias

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Using Affordable LED Arrays for Photo-Stimulation of Neurons

Acessível usando Arrays LED para Foto Estimulação de Neurônios

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

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One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

Um rato, duas culturas: isolamento e cultivo de células-tronco neurais Adulto de as duas zonas neurogênica Individual Ratos

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The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism

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Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

Rápido genotipagem dos animais Seguido por Estabelecer culturas primárias de neurônios do cérebro

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

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Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

Quantificação de Endosome e Moosidades de Lisosoma em Neurônios Cultivados Usando Sondas Fluorescentes

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Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

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Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

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It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...