Trabalho apoiado pelo National Institutes of Health Grants R01DC10146/R01DC010397, NIDCD P30 Núcleo de Pesquisa DC006276 e IES. Seu conteúdo é de responsabilidade exclusiva dos autores e não representam necessariamente a posição oficial do NIH ou IES. Os autores declaram que não há conflito real ou potencial de interesse.

1. Isolamento de CCE <ol><li> Iniciar este procedimento colhendo ossos temporais de cobaias, ratos ou o seu modelo de mamíferos. </li><li> Em seguida, abra os ossos temporais usando uma pinça martelo, a fim de expor a cóclea, e mergulha-as na Leibovitz L-15. Remover o excesso de osso com cuidado, mantendo a casca óssea intacta. Considerando que este é um procedimento geral aplicável aos ossos temporais de qualquer espécie de mamíferos, pequenas mudanças com a técnica pode ser necessária quando se lida com ossos de animais muito pequenos. Em animais velhos a bula é geralmente calcificada, introduzindo uma complicação adicional ao procedimento. </li><li> Sob observação microscópica, abrir a região apical da cóclea e remover estria vascular e ligamento espiral com a ponta de uma lâmina de bisturi # 11, um ponto de micro e pegar uma pinça fina. </li><li> Leve o Órgão de Corti fora do modíolo coclear usando a pinça, e colocá-lo em colagenase 1mg/ml em L-15 em temperatura ambiente por 5 min. </li><li> Se CCEs do giro basal da cóclea são necessários, retire a casca óssea cobrindo a base da cóclea com a picareta, e separar a espiral a partir do osso temporal usando a lâmina de bisturi antes de remover o órgão de Corti. </li><li> Transferir o órgão de Corti para a câmara de gravação usando uma seringa de 50 mL Hamilton. Depois, as células se dissociam pelo refluxo através da agulha. </li></ol> 2. Instalação Experimental <ol><li> Diagrama do gerador de campo elétrico externo alternada (EAEF) e suas ligações com o sistema de captação de imagem (Fig. 1). O circuito de controle foi especialmente implementado no Núcleo de Engenharia do House Ear Institute. </li><li> A configuração experimental utilizada em nossos experimentos consiste de um microscópio 135TV Axiovert invertido (Zeiss, Thornwood, NY) com um sistema de iluminação alternativa baseada em LED (High Power LED System-36AD3500, Lightspeed Technologies, Campbell, CA), dois micromanipuladores eletrônico (Eppendorf &quot;PatchMan&quot;, Alemanha), um PC ultra-controlado de alta velocidade Photron FASTCAM X 1024 PCI da câmera (Photron EUA Inc.) no porto Keller e uma câmera CCD adicionais regulares no porto trinocular. A câmera FASTCAM é capaz de capturar imagens em altas freqüências (até 100.000 fps) e alta resolução (por exemplo, 1024 x 1024 pixels a 1000 fps, 512 x 128, 10.000 fps, 384 x 96 em 18.000 fps, e assim por diante). As imagens fornecidas pela câmera de alta velocidade são diretamente observados no monitor de PC, enquanto a câmera CCD está conectado a um monitor diferente. O sistema de iluminação baseada em LED funciona em dois modos diferentes: analógico de baixa potência e alta potência digital. Todos os procedimentos preliminares (posicionamento de eletrodos, posicionamento de célula, foco, etc) são realizadas usando o modo de baixa potência analógico. A iluminação de alta potência é ligado pela abertura do obturador da câmera e, então, desligado pelo fechamento do obturador, facilitando a dissipação de calor. Software caseiro, também desenvolvido no Instituto House Ear Núcleo de Engenharia, em execução no PC mesmo controla o disparo da câmera de alta velocidade, o sistema de iluminação baseados em LEDs, e as EAEF. Uma câmera de foto digital convencional na porta frontal permite ainda os quadros conforme necessário. (Fig. 2 A). </li><li> Eletrodos (dois fios 0,25 mm de diâmetro-Ag com a distância da ponta de 0,8 mm) são levados a posição usando um dos micromanipuladores eletrônicos. Posição eletrodos é monitorada visualmente e por meio da imagem microscópica; uma mudança no plano focal da imagem indica que os eletrodos tocou o fundo da câmara experimental. Inicialmente, o campo elétrico é calibrado usando um eletrodo externo. Este eletrodo mede o potencial elétrico em pontos diferentes, gerando um &quot;mapa&quot; do campo elétrico. Se uma célula do cabelo isolado exterior é colocado entre as pontas dos eletrodos com o seu paralelo eixo longitudinal ao EAEF aplicado, ele se moverá alongamento e encurtamento na mesma freqüência do campo elétrico. Se a célula é colocado perpendicular ao campo um tipo diferente de resposta OHC (flexão) podem ser observados e investigados. (Fig. 2 B) </li></ol> 3. EAEF estimulação e captação de imagem <ol><li> Quatro diferentes protocolos de estimulação são selecionáveis ​​(Fig. 3): <ol><li> freqüência única e contínua (Fig. 3 A). Note-se que tipo de modo de estímulo, freqüência, amplitude e onda pode ser selecionado usando o home-made software de controle (círculos vermelhos). </li><li> única freqüência bursted (Fig. 3 B). O comprimento das explosões e as lacunas entre estourou também são selecionáveis. </li><li> linear de varredura (Fig. 3 C). As freqüências inicial e final são selecionáveis. </li><li> múltiplas-estimulação (Fig. 3 D). Freqüência única e varre linear podem ser combinados em um único experimento. Depois de selecionar os parâmetros correspondentes, o software de controle e configura o sistema permite ao operador iniciar-luz de gravação de vídeo sincronizados e estimulação de células, clicando em um único botão notela do computador. </li></ol></li><li> As imagens são capturadas em formato AVI, para posterior análise em altas freqüências. </li></ol> 4. Resultados representante <ol><li> Neste filme, dois isolados células ciliadas externas mostram mudanças no comprimento ou curvatura quando estão sendo estimuladas com um campo elétrico externo alternada que é paralela ou transversal, respectivamente, ao seu eixo longitudinal. (Filme # 2). </li><li> Respostas CCE móveis são analisados ​​off-line utilizando software ProAnalyst (Xcitex Inc., Cambridge, MA). &quot;Feature Tracking&quot; função neste software fornece a distância entre dois pontos de quadro a quadro (Fig. 4). Durante célula encurtando a distância entre os pontos selecionados no (placa cuticular; cor vermelha) ápice ea base da célula (pólo Basal; cor verde) é menor, e aumenta com o alongamento das células. O filme mostra o software análise quadro a quadro-a mudanças no comprimento. O painel na parte inferior da imagem mostra o traço do movimento. Neste exemplo, a variação total de comprimento é de cerca de 6,5 pixels. </li><li> &quot;Contour Tracking&quot; função no software ProAnalyst pode detectar automaticamente borda da célula e medir a área da secção óptica (Fig. 5). </li><li> Microesferas de poliestireno adicionado à solução de banho de forma aleatória e com firmeza anexar à membrana plasmática (Fig. 6 A). Microesferas diferentes podem ser selecionados simultaneamente, eo software pode rastrear automaticamente todos eles quadro a quadro. Desta forma, as células podem ser divididos em seções e da motilidade de cada seção avaliada de forma independente. (Fig. 6 A) </li><li> Ao selecionar microesferas localizados nas bordas laterais da imagem celular, mudanças no comprimento de cada segmento e as mudanças no ângulo de um segmento que diz respeito a outros (flexão) também pode ser avaliada de forma independente. (Fig. 6B) </li></ol><img src="/files/ftp_upload/2965/2965fig1.jpg" alt="Figura 1"/> Figura 1. Diagrama do gerador EAEF e suas ligações com o sistema de captura de imagem. <img src="/files/ftp_upload/2965/2965fig2.jpg" alt="Figura 2"/> Figura 2. Imagem A) da instalação experimental. B) Detalhe da etapa de microscópio, com charges dos eletrodos e um OHC única colocada entre eles com o seu paralelo eixo longitudinal ao campo elétrico. <img src="/files/ftp_upload/2965/2965fig3.jpg" alt="Figura 3"/> Figura 3. A interface de usuário) do software de controle caseiro configurado para uma única freqüência de estimulação. Os parâmetros selecionados estão circulados em vermelho. B) interface de usuário da home-made software de controle configurado para estourar única freqüência de estimulação. C) interface de usuário da home-made software de controle configurado para a estimulação de varredura linear. D) interface de usuário do software de controle caseiro configurado para estimulação multi. <img src="/files/ftp_upload/2965/2965fig4.jpg" alt="Figura 4"/> Figura 4. Quadro único de um OHC com dois pontos selecionados na base (verde) eo ápice (vermelho) da célula, respectivamente, usando o &quot;recurso de rastreamento&quot; função do software ProAnalyst. A curva abaixo da célula mostra a mudanças periódicas na distância entre os pontos selecionados associados com estimulação elétrica. Movendo a barra vertical frames diferentes podem ser selecionadas para análise individual. <img src="/files/ftp_upload/2965/2965fig5.jpg" alt="Figura 5"/> Figura 5. O &quot;Contour Tracking&quot; em função ProAnalyst detecta a borda da célula e automaticamente medir a área da secção óptica. <img src="/files/ftp_upload/2965/2965fig6.jpg" alt="Figura 6"/> Figura 6. Uma imagem) Capturado de um OHC isoladas decorados com microesferas de poliestireno (superior), ea mesma imagem com cinco microesferas selecionadas individualmente (em baixo). Uma cor diferente foi designado para cada micro, e seus deslocamentos podem ser individualmente e automaticamente quadro a quadro rastreados, analisados ​​e comparados. B) Segmentos de comprimento arbitrário pode ser definido por escolha de poliestireno microesferas localizados nas bordas da célula, e as alterações no comprimento destes segmentos, bem como alterações na orientação de um segmento que diz respeito aos outros (célula dobra) pode ser automaticamente avaliado quadro a quadro com o software de análise de imagem. Filme 1. Isolamento de CCEs cobaia. <a href="http://www.jove.com/files/ftp_upload/2965/2965_Movie1_web.mov">Clique aqui para assistir ao vídeo</a> Filme 2. CCE paralelas e perpendiculares ao EAEF mostrando eletromotilidade típicos e respostas flexão. <a href="http://www.jove.com/files/ftp_upload/2965/2965_Movie2_web.mov">Clique aqui para assistir ao vídeo</a>

<ol>
	<li>Frolenkov, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+insights+into+the+morphogenesis+of+inner+ear+hair+cells.">Genetic insights into the morphogenesis of inner ear hair cells.</a> Nat Rev Genet. 5, 489-498 (2004).</li><li>Ashmore, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+outer+hair+cell+motility.">Cochlear outer hair cell motility.</a> Physiol Rev. 88, 173-210 (2008).</li><li>Dallos, P., Fakler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prestin,+a+new+type+of+motor+protein.">Prestin, a new type of motor protein.</a> Nature Rev. Mol. Cell Biol. 3, 104-111 (2002).</li><li>Frolenkov, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+outer+hair+cell+bending+in+an+external+electrical+field.">Cochlear outer hair cell bending in an external electrical field.</a> Biophys. J. 73, 1665-1672 (1997).</li><li>Matsumoto, N., Kalinec, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+Prestin-Dependent+and+Prestin-Independent+Components+from+Complex+Motile+Responses+in+Guinea+Pig+Outer+Hair+Cells.">Extraction of Prestin-Dependent and Prestin-Independent Components from Complex Motile Responses in Guinea Pig Outer Hair Cells.</a> Biophys J. 89, 4343-4351 (2005).</li><li>Matsumoto, N., Kalinec, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prestin-dependent+and+prestin-independent+motility+of+guinea+pig+outer+hair+cells.">Prestin-dependent and prestin-independent motility of guinea pig outer hair cells.</a> Hear Res. 208, 1-12 (2005).</li><li>Ashmore, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+remarkable+cochlear+amplifier.">The remarkable cochlear amplifier.</a> Hear Res. 266, 1-17 (2010).</li><li>Kitani, R., Kakehata, S., Kalinec, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motile+responses+of+cochlear+outer+hair+cells+stimulated+with+an+alternating+electrical+field.">Motile responses of cochlear outer hair cells stimulated with an alternating electrical field.</a> Hearing Research. , (2011).</li><li>Dallos, P., Evans, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+outer+hair+cell+motility:+corrections+and+addendum.">High-frequency outer hair cell motility: corrections and addendum.</a> Science. 268, 1420-1421 (1995).</li><li>Frank, G., Hemmert, W., Gummer, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limiting+dynamics+of+high-frequency+electromechanical+transduction+in+outer+hair+cells.">Limiting dynamics of high-frequency electromechanical transduction in outer hair cells.</a> Proc. Natl. Acad. Sci. 96, 4420-4425 (1999).</li><li>Santos-Sacchi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+frequency+limit+and+phase+of+outer+hair+cell+motility:+effects+of+the+membrane+filter.">On the frequency limit and phase of outer hair cell motility: effects of the membrane filter.</a> J. Neurosci. 12, 1906-1916 (1992).</li><li>Inou&#233;, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Video Microscopy. , (1986).</li></ol>

Não há conflitos de interesse declarados.

O método experimental aqui apresentado permite estimar respostas OHC móveis na faixa de kHz sem qualquer restrição ao movimento da célula. Protocolos de estimulação diferentes, marcadores adicionais (microesferas), bem como alterações na orientação da célula em relação ao campo elétrico, torná-lo possível investigar novos aspectos da motilidade das CCE, com um nível de detalhes anteriormente inacessíveis. Outros métodos, por exemplo, aqueles que usam fotodiodos 9 ou laser Doppler vibrometr...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Leibovitz L-15 </td><td> Gibco </td><td> 21083 </td><td></td></tr><tr><td> Colagenase (tipo 4) </td><td> Sigma-Aldrich </td><td> C5138 </td><td> 1mg/mL em L-15 </td></tr></tbody></table>

investigating outer hair cell motility with a combination of external alternating electrical field stimulation and high-speed image analysis

CCE são células cilíndricas sensório-motor localizado no órgão de Corti, o órgão auditivo dentro do ouvido dos mamíferos interior. O nome de &quot;células ciliadas&quot; deriva do seu pacote característica apical de estereocílios, um elemento crítico para a detecção e transdução de energia sonora 1. CCE são capazes de mudar de forma-alongado, encurtar e dobre-em resposta à estimulação elétrica, mecânica e química, uma resposta motora considerada crucial para a amplificação coclear dos sinais acústicos 2. Estimulação OHC induz duas diferentes respostas motile: i) eletromotilidade, motilidade rápida aka, mudanças no comprimento na faixa de microssegundos derivados com motor eléctrico mudanças conformacionais em proteínas motoras densamente no OHC membrana plasmática, e ii) a motilidade lenta, muda de forma no milissegundos para segundos gama envolvendo reorganização do citoesqueleto 2, 3. OHC de flexão é associado com eletromotilidade, e resultar tanto de uma distribuição assimétrica de proteínas motoras na membrana plasmática lateral, ou estimulação elétrica assimétrica dessas proteínas motoras (por exemplo, com um campo elétrico perpendicular ao eixo longitudinal das células) 4. Estímulos mecânicos e químicos induzem essencialmente lento respostas móveis, mesmo que as mudanças nas condições iônica das células e / ou seu ambiente também podem estimular a membrana plasmática-embedded do motor proteínas 5, 6. Desde respostas OHC móveis são um componente essencial do amplificador coclear, a análise qualitativa e quantitativa dessas respostas móveis em freqüências acústicas (cerca de 20 Hz a 20 kHz em humanos) é um assunto muito importante no campo da audição investigação 7. O desenvolvimento da nova tecnologia de imagem que combina alta velocidade câmeras de vídeo, sistemas baseados em LEDs de iluminação, e software de análise sofisticada imagem agora fornece a capacidade de executar qualitativa confiável e estudos quantitativos da resposta móveis de CCEs isolado a um campo elétrico externo alternada (EAEF) 8. Esta é uma técnica simples e não invasivo, que contorna a maioria das limitações das abordagens anteriores 9-11. Além disso, o sistema de iluminação baseada em LED proporciona brilho extremo com insignificantes efeitos térmicos nas amostras e, por causa do uso de microscopia de vídeo, resolução óptica é de pelo menos 10 vezes maior do que com as técnicas convencionais de microscopia de luz 12. Por exemplo, com a configuração experimental descrito aqui, mudanças no comprimento das células de cerca de 20 nm pode ser rotineiramente e de forma confiável detectados em freqüências de 10 kHz, e esta resolução pode ser melhorada em freqüências mais baixas. Estamos confiantes de que essa abordagem experimental ajudará a ampliar nossa compreensão dos mecanismos celulares e moleculares subjacentes a motilidade das CCE.

Watch this Scientific Journal Video about Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis at JoVE.com

Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis

Um método confiável para investigar células ciliadas externas (CCE) respostas móveis, incluindo eletromotilidade, motilidade lenta e flexão, é descrito. Motilidade das CCE é eliciada pela estimulação com um campo elétrico externo alternada, eo método tira proveito da alta velocidade de gravação de imagem, baseada em LED, iluminação e de última geração de software de análise de imagem.

Investigando Motilidade das células ciliadas externas com uma combinação de estimulação de campo externo elétrica alternada de alta velocidade e Análise de Imagem

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives ...

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Research

JoVE Journal

Neuroscience

13.4K visualizações.  House Ear Institute. Um método confiável para investigar células ciliadas externas (CCE) respostas móveis, incluindo eletromotilidade, motilidade lenta e flexão, é descrito. Motilidade das CCE é eliciada pela estimulação com um campo elétrico externo alternada, eo método tira proveito da alta velocidade de gravação de imagem, baseada em LED, iluminação e de última geração de software de análise de imagem.

Vídeo: Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS: Usando o protocolo Theta-Burst para Explorar Mechasnism de Plasticidade em Indivíduos com Síndrome do X Frágil e Autismo

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

Neurociência

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

Visualização e manipulação genética de dendritos e espinhas no córtex cerebral e hipocampo do rato usando<em&gt; In utero</em&gt; Eletroporação

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

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 ...

A Human-machine-interface Integrating Low-cost Sensors with a Neuromuscular Electrical Stimulation System for Post-stroke Balance Rehabilitation

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing delivery of oxygen and nutrients. About half of the stroke survivors are left with some degree of disability. Innovative methodologies for restorative neurorehabilitation are urgently required to reduce long-term disability. The ability of the nervous system to reorganize its structure, function and connections as a response to intrinsic or extrinsic stimuli is called neuroplasticity. Neuroplasticity is involved in post-stroke functional disturbances, but also in rehabilitation. Beneficial neuroplastic changes may be facilitated with non-invasive electrotherapy, such as neuromuscular electrical stimulation (NMES) and sensory electrical stimulation (SES). NMES involves coordinated electrical stimulation of motor nerves and muscles to activate them with continuous short pulses of electrical current while SES involves stimulation of sensory nerves with electrical current resulting in sensations that vary from barely perceivable to highly unpleasant. Here, active cortical participation in rehabilitation procedures may be facilitated by driving the non-invasive electrotherapy with biosignals (electromyogram (EMG), electroencephalogram (EEG), electrooculogram (EOG)) that represent simultaneous active perception and volitional effort. To achieve this in a resource-poor setting, e.g., in low- and middle-income countries, we present a low-cost human-machine-interface (HMI) by leveraging recent advances in off-the-shelf video game sensor technology. In this paper, we discuss the open-source software interface that integrates low-cost off-the-shelf sensors for visual-auditory biofeedback with non-invasive electrotherapy to assist postural control during balance rehabilitation. We demonstrate the proof-of-concept on healthy volunteers.

A novel low-cost human-machine interface for interactive post-stroke balance rehabilitation system is presented in this article. The system integrates off-the-shelf low-cost sensors towards volitionally driven electrotherapy paradigm. The proof-of-concept software interface is demonstrated on healthy volunteers.

A-máquina interface humana Integrando sensores de baixo custo com uma estimulação do sistema neuromuscular eléctrico para pós-AVC Reabilitação Balance

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing ...

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

Avaliação da função neuromuscular Usando percutânea Estimulação Elétrica Nervosa

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

Vídeo Imagem e Mapas espaço-temporais para analisar Motilidade Gastrointestinal em Ratos

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific frequency and in turn modulate ongoing cortical oscillatory activity. This neurotool allows the establishment of a causal link between endogenous oscillatory activity and behavior. Most of the tACS studies have shown online effects of tACS. However, little is known about the underlying action mechanisms of this technique because of the AC-induced artifacts on Electroencephalography (EEG) signals. Here we show a unique approach to investigate online physiological frequency-specific effects of tACS of the primary motor cortex (M1) by using single pulse Transcranial Magnetic Stimulation (TMS) to probe cortical excitability changes. In our setup, the TMS coil is placed over the tACS electrode while Motor Evoked Potentials (MEPs) are collected to test the effects of the ongoing M1-tACS. So far, this approach has mainly been used to study the visual and motor systems. However, the current tACS-TMS setup can pave the way for future investigations of cognitive functions. Therefore, we provide a step-by-step manual and video guidelines for the procedure.

Transcranial Alternating Current Stimulation (tACS) allows the modulation of cortical excitability in a frequency-specific fashion. Here we show a unique approach which combines online tACS with single pulse Transcranial Magnetic Stimulation (TMS) in order to "probe" cortical excitability by means of Motor Evoked Potentials.

Efeitos da estimulação da corrente alternada transcraniana no córtex Motor primário por on-line abordagem combinada com estimulação magnética transcraniana

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...

Transcranial Electrical Brain Stimulation in Alert Rodents

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in humans is transcranial direct current stimulation (tDCS). Less frequently, transcranial alternating current stimulation (tACS) or transcranial random noise stimulation (tRNS), a specific form of tACS using an electrical current applied randomly within a pre-defined frequency range, is used. The increase of noninvasive electrical brain stimulation research in humans, both for experimental and clinical purposes, has yielded an increased need for basic, mechanistic, safety studies in animals. This article describes a model for transcranial electrical brain stimulation (tES) through the intact skull targeting the motor system in alert rodents. The protocol provides step-by-step instructions for the surgical set-up of a permanent epicranial electrode socket combined with an implanted counter electrode on the chest. By placing a stimulation electrode into the epicranial socket, different electrical stimulation types, comparable to tDCS, tACS, and tRNS in humans, can be delivered. Moreover, the practical steps for tES in alert rodents are introduced. The applied current density, stimulation duration, and stimulation type may be chosen depending on the experimental needs. The caveats, advantages, and disadvantages of this set-up are discussed, as well as safety and tolerability aspects.

This protocol describes a surgical set-up for a permanent epicranial electrode socket and an implanted chest electrode in rodents. By placing a second electrode into the socket, different types of transcranial electrical brain stimulation can be delivered to the motor system in alert animals through the intact skull.

Estimulação transcraniana de elétrica cerebral em roedores alertas

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Estimulação elétrica transcraniana crônica e gravação Intracortical em ratos

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in 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 ...