Name: gold nanorod-assisted optical stimulation of neuronal cells
Uploaded: 2015-04-27T14:00:00.000+00:00
Duration: 9 min 31 s
Description: Watch this Scientific Journal Video about Gold Nanorod-assisted Optical Stimulation of Neuronal Cells at JoVE.com

Os autores gostariam de agradecer NanoVentures Austrália por apoio de financiamento de viagens e Prof. John Haycock por ter sido sede parcialmente esta pesquisa da Universidade de Sheffield e Ms. Jaimee Mayne por sua ajuda durante as filmagens.

1. Au NRs Preparação Nota: Au NRs pode ser sintetizado por uma série de receitas 16, ou adquiridos de fornecedores comerciais. <ol><li> Medir a densidade óptica inicial (OD) da solução de Au NR via espectroscopia de UV-Vis, por um registo dos valores de absorção de 300 nm a 1000 nm, com uma resolução de 0,5-2 nm. Fazer variar o volume da solução a ser usado com a cuvete disponível. </li><li> Avaliar a concentração molar inicial NP com uma técnica de 17 adequada (por exemplo, UV-Vis, única partícula espectrometria de massa com plasma indutivamente acoplado, microscopia eletrônica de transmissão) ou usar os valores de concentração fornecidos pelo fornecedor. </li><li> Prepara-se uma solução estoque de 5 ml por diluição da amostra inicial de Au NR para atingir uma DO = 1. Para a melhor repetibilidade, manter constante o OD para todas as amostras testadas. Seguir o protocolo do vendedor comercial para a composição do solvente de diluição. Se não tiver certeza, Use água deionizada. </li><li> Centrifugar 1 ml da solução de Au NR duas vezes durante 15 min a 7800 xg para remover qualquer excesso de produto químico a partir da solução. Ciclos de centrifugação podem variar em vigor e tempo (por exemplo, 20 min a 5.600 x g) 18. </li><li> Remover o sobrenadante e re-suspender as NRs em água deionizada. Como partículas re-suspensão pode formar agregados em solução, prepará-los diariamente para ter os melhores resultados. Alternativamente, armazenar em um frigorífico para não mais do que uma semana. Não congelar. </li><li> Antes de utilizar, sob condições de cultura de células, sonicar a solução Au NR durante 5 min e, em seguida esterilizar com luz UV durante 30 minutos (intensidade da radiação UV não menos do que 400 mW ∙ m -2 a 254 nm). </li></ol> 2. NG108-15 celular neuronal Linha Cultura e Diferenciação <ol><li> Para o meio de cultura de células, preparar 500 ml de meio estéril de Dulbecco modificado por Eagle (DMEM) contendo 10% (w / v) de soro fetal de vitelo (FCS), 1% (w / v) de L-glutamina, 1%(W / v) de penicilina / estreptomicina e 0,5% (w / v) de anfotericina B. Nota: Os suplementos podem ser aliquotado, armazenado a -20 ° C e adicionado ao meio no dia necessária. Meio de cultura celular pode ser refrigerado numa condição estéril durante um período máximo de 1 mês. </li><li> Para o meio de diferenciação celular, preparar 50 ml de DMEM estéril contendo 1% (w / v) de L-glutamina, 1% (w / v) de penicilina / estreptomicina e 0,5% (w / v) de anfotericina B. </li><li> Cresça NG108-15 células neuronais em 10 ml de meio de cultura de células em frascos T75 em poliestireno numa incubadora com uma atmosfera humidificada (5% de CO2 a 37 ° C). Normalmente, sementes 1,5-2 × 10 5 células em cada frasco para estar pronto em 3-4 dias. Mudar meio de cultura celular a cada dois dias. Nota: Para evitar desvios genéticos ou variação, não use as células mais velhas do que a passagem de 21 para experimentos. </li><li> Quando 70-80% confluentes em cultura, mudar o meio com meio de cultura de células frescas quente. Mecanicamente separar as células por suavemente KNOCrei o fundo do frasco confluente. Não use tripsina. </li><li> Centrifugar a suspensão de células durante 5 min a 600 xg e re-suspender o sedimento celular em 2 ml de meio de diferenciação da célula quente. </li><li> Semente 2 × 10 4 células / cm2 em uma cultura de tecidos de poliestireno placa de 96 poços com 200 ul de meio de diferenciação celular. Incubar a experiência durante 1 dia a 5% de CO 2/37 ° C. </li><li> Adicionar entre 3,2 × 10 × 1 -4,2 9 0 10 partículas / ml de solução Au NR no dia 2 e incubar por 24 horas adicionais. Não adicione as partículas para os experimentos de controle. Observação: Como um controlo alternativo, Au PN com um pico de absorção de comprimento de onda bem diferenciado pode ser utilizado para fins de comparação 14. Para um comportamento celular mais consistente, manter a densidade de células constante e não modificar a superfície bem antes da semeadura de células. </li></ol> 3. Aperfeiçoamento crescimento de neurites <ol><li> Casalo laser com uma fibra óptica monomodo (abertura numérica = 0,13) e terminá-lo com um conector de fibra (FC conectores são convenientes e comumente disponíveis). Medir a potência do laser de saída com um medidor de potência padrão. Para obter os resultados mais eficazes, corresponder ao comprimento de onda de pico do laser para o pico de ressonância de plasmon de NRs. </li><li> No dia 3 após a incubação NR, fixar o conector FC para o bem. Irradiar as amostras e os controlos em TA durante 1 min em onda contínua, para diferentes potências de laser. Permitir que a cultura prosseguir durante mais 3 dias em 5% de CO 2/37 ° C. Repetir a irradiação com laser durante um mínimo de 3 medições independentes. Nota: Diferentes tempos de irradiação e frequências de pulsação pode ser seleccionado, dependendo da aplicação. </li><li> Caracterizar o laser em termos de diâmetro do feixe e irradiação laser (W ∙ cm -2). <ol><li> Para uma fibra monomodo padrão, use NA = n ∙ sin θ,onde n é o índice de refracção do meio em utilização e θ é a metade do ângulo do cone da luz que sai da fibra (ver Figura 1A). De trigonometria, r = tan θ ∙ d, em que r é o raio do feixe e d é a distância entre a fibra e a amostra. O conector FC coincide com o diâmetro do poço, por conseguinte, iluminando a amostra a uma distância d = 2,70 ± 0,20 mm. Luz sai da fibra em água (n = 1,33), dando r = tan (sin-1 (NA / n)) ∙ d. </li><li> Usando esta última equação, calcular o raio do feixe laser, o feixe de área correspondente e a irradiação a laser média (potência do laser dividida pela área do feixe) para o alvo (ver o gráfico de exemplo na Figura 1B). Estes valores representam a irradiância média sobre a área iluminada. </li><li> Avaliar os erros para as medições com a teoria geral da propagação de erros. Nota: A distância bntre o conector FC e a amostra pode ser medida por fotografias do arranjo experimental e de pós-processamento da imagem utilizando software apropriado (por exemplo ImageJ). </li></ol></li><li> No dia 5, remover o meio de diferenciação de células a partir das experiências e fixar as amostras com 3,7% (v / v) de solução de formaldeído, durante 10 min, em seguida, permeabilizar as células com 0,1% (v / v) de Triton X-100 durante 20 min. </li><li> Adicionar 3% (w / v) de albumina de soro bovino (BSA) para as amostras durante 60 minutos para bloquear os locais de ligação da proteína que não reagiu. Rotular as amostras durante a noite para anti-βIII-tubulina (5 ug / ml em PBS suplementado com 1% de BSA) a 4 ° C. </li><li> Incubar as células durante 90 min no escuro com um anticorpo secundário apropriado (anticorpo de ratinho anti-TRITC por exemplo conjugado de IgG) utilizando uma concentração de 0,4-2 ug / ml em 1% de BSA em PBS. Rotular os núcleos celulares com DAPI (0,1 ug / ml em água desionizada) durante 10 min. Nota: conjugação de anticorpos e mig concentraçãoht variar de acordo com o protocolo da empresa. Lavar as amostras duas vezes com PBS durante 5 min após cada etapa de coloração. <img alt="Figura 1" src="/files/ftp_upload/52566/52566fig1.jpg" /></li><li> Amostras de imagem por epifluorescência ou microscopia confocal, utilizando pelo menos um 20 × objetivo. Escolha do microscópio filtra em conformidade com o anticorpo secundário. Selecione um filtro de DAPI (λ EX = 358 nm; λ EM = 488 nm) para visualizar os núcleos celulares. </li><li> Analisar as imagens por avaliar: i) o comprimento de neurites máximo (ficha o comprimento da ponta da neurites para o início do corpo celular), ii) o número de neurites por célula neuronal (resumir todas as neurites por célula) e iii) a percentagem de células com neurites (dividindo o número total de células que expressam βIII-tubulina pelo número total de células com um núcleo corou positivamente) 19. </li></ol> 4. laser-induzida intracelular de Ca2 + de imagem <ol&gt; <li> Prepara-se uma a 20% (w / v) de solução de estoque de Pluronic F-127 por dissolução de 2 g de soluto em 10 ml de dimetilsulfóxido anidro (DMSO). Aquece-se a solução de Pluronic F-127 a 40 ° C durante cerca de 20 min para aumentar a solubilidade. Prepare com antecedência se armazenado à temperatura ambiente. </li><li> No dia 3 seguinte NR incubação, preparar uma solução de sal equilibrada (BSS; NaCl 135 mM, KCl 4,5 mM, CaCl 2 1,5 mM, MgCl 2 0,5 mM, glicose 5,6 mM, e 10 mM de HEPES, pH 7,4) e complementado com 5 ? M de Fluo-4 AM durante DMSO e 0,1% (w / v) de Pluronic F-127 solução. Nota: solução de BSS pode ser preparado antecipadamente e refrigerados durante um período máximo de 1 mês. Adicione os suplementos (Fluo-04:00 e Pluronic F-127) no dia do experimento. </li><li> Remova o meio de diferenciação celular e substituí-lo com a solução de BSS suplementado. Para obter os melhores resultados com um microscópio confocal invertido, incubar as células em um micro-poços da lâmina. </li><li> Carga NG108-15 células com Fluo-04:00durante 30 min no escuro a 5% de CO 2/37 ° C. Após o tempo de incubação, as amostras lavar duas vezes com BSS para remover qualquer fluoróforo descarregado extracelular. </li><li> Pares a laser com uma fibra óptica de modo simples e clivar a ponta, utilizando técnicas convencionais. Observar a ponta resultante sob um microscópio óptico, para assegurar uma superfície de alta qualidade (ou seja, a ponta deve ser perpendicular ao eixo da fibra e plana sobre a inspecção microscopia). <ol><li> Medir a potência do laser de saída com um medidor de potência padrão. Insira o fibra entrega a luz em um suporte de fibra óptica e afixá-lo a um microposicionador. Consulte Brown et al. 20 para obter mais detalhes sobre a preparação de fibra óptica e de posicionamento. Atenção: Siga as regras gerais de segurança do laser durante a medição da potência do laser (por exemplo, não olhe diretamente para o feixe de laser, usar óculos de segurança a laser durante o manuseio, evitar a exposição a luz difusa para outros usuários de laboratório) &lt;./ Li&gt; </ol></li><li> Ligue um osciloscópio para o laser portátil para monitorar a modulação óptica. Use um sinal binário com freqüências variáveis ​​(0,5-2 Hz) e comprimentos de pulso (20-100 ms). Ligue o laser utilizando o sinal de modulação como a lógica transistor-transistor (TTL) de entrada para o microscópio, após a configuração mostrada na Paviolo et al. 13. <ol><li> Colocar as células sob um microscópio confocal invertido e posicionar a fibra de entrega de luz 250 ± 50 mm de distância a partir da célula alvo em modo de iluminação de transmissão. A esta distância, utilizar as equações apresentadas em 3.3.1 para calcular o raio do feixe para o alvo. </li></ol></li><li> Usar um laser de árgon-ião (488 nm) para excitar o corante Fluo-4 AM internalizado (λ EX = 494 nm; λ EM = 516 nm) e o LD sincronizado para a excitação de NRs endocitose. Realize a imagem de TR de amostras e controles usando pelo menos um objectivo de 40 ×, recolhendo scans de séries temporaiscom 256 × 256 pixels / resolução de quadro no modo de ida e volta, com uma frequência de pelo menos 4 Hz. Realizar uma gravação sem qualquer excitação LD para identificar qualquer interferência com laser de argônio-ion (ruído da linha de base). </li><li> Retire o fundo a partir dos dados utilizando adaptativa ponderados penalizados mínimos quadrados algoritmos 21. Traçar as variações induzidas Ca2 + como uma função do tempo. Analisar os picos transientes na intensidade de fluorescência, utilizando software de imagem pós-processamento por thresholding a um nível de três vezes o desvio padrão do ruído de linha de base (3σ). </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+subacute+and+chronic+toxicity+of+cetyltrimethylammonium+bromide+(CTAB),+a+cationic+surfactant,+in+the+rat.">The subacute and chronic toxicity of cetyltrimethylammonium bromide (CTAB), a cationic surfactant, in the rat.</a> Arch. Toxicol. 35 (2), 91-96 (1976).</li><li>Juste, J., Pastoriza-Santos, I., Liz-Marz&#225;n, L., Mulvaney, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanorods:+synthesis,+characterization+and+applications.">Gold nanorods: synthesis, characterization and applications.</a> Coord. Chem. Rev. 249 (17-18), 1870-1901 (2005).</li><li>Dreaden, E. C., Alkilany, A. M., Huang, X., Murphy, C. J., El-Sayed, 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=The+golden+age:+gold+nanoparticles+for+biomedicine.">The golden age: gold nanoparticles for biomedicine.</a> Chem. Soc. Rev. 41 (7), 2740-2779 (2012).</li><li>Huang, X., Jain, P. K., El-Sayed, I. H., El-Sayed, 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=Plasmonic+photothermal+therapy+(PPTT)+using+gold+nanoparticles.">Plasmonic photothermal therapy (PPTT) using gold nanoparticles.</a> Lasers Med. Sci. 23 (3), 217-228 (2008).</li><li>Albert, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPV4+channels+mediate+the+infrared+laser-evoked+response+in+sensory+neurons.">TRPV4 channels mediate the infrared laser-evoked response in sensory neurons.</a> J. Neurophysiol. 107 (12), 3227-3234 (2012).</li><li>Garcia-Elias, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol-4,5-biphosphate-dependent+rearrangement+of+TRPV4+cytosolic+tails+enables+channel+activation+by+physiological+stimuli.">Phosphatidylinositol-4,5-biphosphate-dependent rearrangement of TRPV4 cytosolic tails enables channel activation by physiological stimuli.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (23), 9553-9558 (2013).</li><li>Shapiro, M. G., Homma, K., Villarreal, S., Richter, C. -. P., Bezanilla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infrared+light+excites+cells+by+changing+their+electrical+capacitance.">Infrared light excites cells by changing their electrical capacitance.</a> Nat. Commun. 3 (736), (2012).</li><li>Roggan, A., Friebel, M., D&#246;rschel, K., Hahn, A., M&#252;ller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+properties+of+circulating+human+blood+in+the+wavelength+range+400-2500+nm.">Optical properties of circulating human blood in the wavelength range 400-2500 nm.</a> J. Biomed. Opt. 4 (1), 36-46 (1999).</li><li>Byrnes, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+promotes+regeneration+and+functional+recovery+and+alters+the+immune+response+after+spinal+cord+injury.">Light promotes regeneration and functional recovery and alters the immune response after spinal cord injury.</a> Lasers Surg. Med. 36 (3), 171-185 (2005).</li><li>Wu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=810+nm+wavelength+light:+an+effective+therapy+for+transected+or+contused+rat+spinal+cord.">810 nm wavelength light: an effective therapy for transected or contused rat spinal cord.</a> Lasers Surg. Med. 41 (1), 36-41 (2009).</li><li>Grossman, N., Schneid, N., Reuveni, H., Halevy, S., Lubart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=780+nm+low+power+diode+laser+irradiation+stimulates+proliferation+of+keratinocyte+cultures:+involvement+of+reactive+oxygen+species.">780 nm low power diode laser irradiation stimulates proliferation of keratinocyte cultures: involvement of reactive oxygen species.</a> Lasers Surg. Med. 22 (4), 212-218 (1998).</li><li>Wong-Riley, M. T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photobiomodulation+directly+benefits+primary+neurons+functionally+inactivated+by+toxins+-+Role+of+cytochrome+c+oxidase.">Photobiomodulation directly benefits primary neurons functionally inactivated by toxins - Role of cytochrome c oxidase.</a> J. Biol. Chem. 280 (6), 4761-4771 (2005).</li><li>Beauvoit, B., Kitai, T., Chance, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+mitochondrial+compartment+to+the+optical+properties+pf+the+rat+liver:+a+theoretical+and+practical+approach.">Contribution of the mitochondrial compartment to the optical properties pf the rat liver: a theoretical and practical approach.</a> Biophys. J. 67 (6), 2501-2510 (1994).</li></ol>

The authors have no competing interests to disclose.

Os protocolos descritos nesta apresentação descrevem como cultura, diferenciar e opticamente estimular as células neuronais usando absorventes extrínsecos. As características NR (por exemplo, dimensões, forma, comprimento de onda de ressonância de plasmon de superfície e química) e os parâmetros de estimulação do laser (comprimento de onda, tais como, duração dos impulsos, taxa de repetição, etc.) pode ser variada para corresponder a diferentes necessidades experimentais. Os efeitos sob...

Estudos recentes têm demonstrado que o aquecimento transitória associada com a absorção de luz infravermelha por água (comprimento de onda&gt; 1400 nm) pode ser utilizado para induzir potenciais de acção em tecidos nervosos 1 e transientes de cálcio intracelular em cardiomiócitos 2. A utilização de luz infravermelha tem suscitado grande interesse para aplicações em próteses neurais, devido ao potencial de resolução espacial mais fina, a falta de um contacto directo com o tecido, a minimização de artefactos de estimulação, e remoção da necessidade de modificar geneticamente as células antes da estimulação ( conforme exigido no optogenética) 1. Apesar de todas estas vantagens, os modelos térmicos desenvolvidos recentemente sugerido que os tecidos / células-alvo pode ser afectada por efeitos cumulativos de aquecimento, quando vários sítios de estímulo e / ou elevadas taxas de repetição são utilizados 3,4. Em resposta a estes desafios, os pesquisadores têm reconhecido o potencial de usar unidade de absorvância extrínsecabros para a estimulação do nervo para produzir efeitos de aquecimento mais localizadas no tecido. Huang et al. Demonstrou esse princípio usando nanopartículas de ferrita superparamagnéticos para ativar remotamente os canais TRPV1 sensíveis à temperatura em células HEK 293 com um campo magnético 5 de rádio-freqüência. Embora esta técnica pode permitir a penetração mais profunda (campos magnéticos relativamente interagem fracamente com o tecido), apenas as respostas foram registadas ao longo de períodos de segundos, em vez de as durações milissegundo necessários em dispositivos biônicos 5. Da mesma forma, Farah et al. Estimulação elétrica demonstrada de neurônios corticais de ratos com micro-partículas negras em vitro. Eles mostraram precisão em nível celular em estimulação usando durações de pulso na ordem de centenas de ms e energias na faixa de μJ, potencialmente permitindo que as taxas de repetência mais rápidos 6. A utilização de absorventes extrínsecos também foi aplicado para induziralterações morfológicas in vitro. Ciofani et al. Mostrou um aumento de ~ 40% no crescimento de células neuronais usando nanotubos de nitreto de boro piezoelétricos excitados por ultra-som 7. Da mesma forma, nanopartículas de óxido de ferro endocitose nas células PC12 têm sido relatados para aumentar a diferenciação de neurites de uma forma dependente da dose, devido à activação de moléculas de adesão celular, com o óxido de ferro 8. Recentemente, o interesse em absorvedores extrínsecos para ajudar a estimulação neural também centrou-se na utilização de nanopartículas de ouro (Au NPS). Au NPs têm a capacidade de absorver a luz laser de forma eficiente no pico plasmónico e dissipar para o ambiente circundante em forma de calor 9. Entre todas as formas de partículas disponíveis, a absorção óptica do nanorods ouro (Au NRs) corresponde convenientemente a janela terapêutica de tecidos biológicos (infravermelho próximo - NIR, comprimento de onda entre 750-1,400 nm) 10. Além disso, no context de estimulação neural, o uso de Au NRs proporciona biocompatibilidade relativamente favoráveis ​​e uma grande variedade de opções de funcionalização de superfície 11. Estudos recentes têm mostrado que um efeito estimulador sobre a diferenciação pode ser induzida após exposições a laser contínua de Au NRs NG 108-15 em células neuronais 12. Da mesma forma, os transientes de cálcio intracelular foram registrados em células neuronais cultivadas com Au NRs após a irradiação com laser modulado com freqüências variáveis ​​e pulso comprimentos 13. Despolarização da membrana celular também foi registada após iluminação por laser NIR de Au NRs em culturas primárias de neurónios do gânglio da espiral 14. A primeira aplicação in vivo com irradiado Au NRs foi demonstrado recentemente. Eom e colegas de trabalho expostos Au NRs em seu pico plasmonic e registrou um aumento de seis vezes na amplitude do potencial de ação do nervo composto (CNaPS) e uma diminuição de três vezes no limiar de estimulação no nervo ciático de ratos. O enresposta hanced foi atribuído a efeitos de aquecimento local que resultam da excitação do pico plasmónico NR 15. No presente trabalho, protocolos para investigar os efeitos da estimulação do laser em NG108-15 células neuronais cultivadas com Au NRs são especificados. Esses métodos fornecem um simples, mas poderoso, caminho para irradiar populações de células in vitro utilizando técnicas de biologia padrão e materiais. O protocolo baseia-se em um diodo laser acoplada a fibra (LD) que permite um funcionamento seguro e repetitivo alinhamento. As Au NR preparação de amostras e métodos de irradiação a laser pode ser alargada a diferentes formas de partículas e culturas de células neuronais, proporcionando que os protocolos de síntese e de cultura específicos são conhecidos, respectivamente.

<table><tbody><tr><td>Au NR</td><td>Sigma Aldrich</td><td>716812</td><td></td></tr><tr><td>NG108-15</td><td>Sigma Aldrich</td><td>8811230</td><td></td></tr><tr><td>DMEM</td><td>Sigma Aldrich</td><td>D6546</td><td></td></tr><tr><td>FCS</td><td>Life Technologies</td><td>10100147</td><td></td></tr><tr><td>L-glutamine</td><td>Sigma Aldrich</td><td>G7513</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>Amphotericin B</td><td>Life Technologies</td><td>15290018</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma Aldrich</td><td>F8775</td><td></td></tr><tr><td>Triton X-100</td><td>BDH</td><td>T8532</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A2058</td><td></td></tr><tr><td>Anti-&amp;beta;III-tubulin</td><td>Promega</td><td>G7121</td><td></td></tr><tr><td>TRITC-conjugated anti-mouse IgG antibody</td><td>Sigma Aldrich</td><td>T5393</td><td></td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>D1306</td><td></td></tr><tr><td>Fluo-4 AM</td><td>Invitrogen</td><td>F14201</td><td></td></tr><tr><td>DMSO</td><td>Sigma Aldrich</td><td>472301</td><td></td></tr><tr><td>Pluronic F-127</td><td>Invitrogen</td><td>P6867</td><td></td></tr><tr><td>UV-Vis spectrometer</td><td>Varian Medical Systems Inc.</td><td>Cary 50 Bio</td><td></td></tr><tr><td>Mini centrifuge</td><td>Eppendorf</td><td>Mini Spin</td><td></td></tr><tr><td>Sonic bath</td><td>Unisonics Australia</td><td>FPX 10D</td><td></td></tr><tr><td>Cell culture incubator</td><td>Kendro</td><td>Hera Cell 150</td><td></td></tr><tr><td>Cell culture centrifuge</td><td>Hettich</td><td>Rotofix 32A</td><td></td></tr><tr><td>Laser diode</td><td>Optotech</td><td>780 nm single mode fibre - coupled LD</td><td></td></tr><tr><td>Optical fiber</td><td>Thorlabs</td><td>780 HP</td><td></td></tr><tr><td>Power meter</td><td>Coherent</td><td>Laser Check</td><td></td></tr><tr><td>ImageJ</td><td>http://rsb.info.nih.gov/ij/index.html</td><td></td><td></td></tr><tr><td>Epifluorescent microscope</td><td>Axon Instruments</td><td>ImageX-press 5000A</td><td></td></tr><tr><td>&amp;mu;-slide well</td><td>Ibidi</td><td>80826</td><td></td></tr><tr><td>Inverted confocal microscope</td><td>Carl Zeiss Microscopy Ltd.</td><td>LSM 510 meta-confocal microscope</td><td></td></tr><tr><td>Oscilloscope</td><td>Tektronix</td><td>TDS210</td><td></td></tr></tbody></table>

gold nanorod-assisted optical stimulation of neuronal cells

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared light by water in neuronal tissue. This technique holds great potential for replacing or complementing standard stimulation techniques, due to the potential for increased localization of the stimulus and minimization of mechanical contact with the tissue. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Moreover, thermal modelling suggests that cumulative heating effects might be potentially hazardous when multiple stimulus sites or high laser repetition rates are used. The protocol outlined below describes an enhanced approach to the infrared stimulation of neuronal cells. The underlying mechanism is based on the transient heating associated with the optical absorption of gold nanorods, which can cause triggering of neuronal cell differentiation and increased levels of intracellular calcium activity. These results demonstrate that nanoparticle absorbers can enhance and/or replace the process of infrared neural stimulation based on water absorption, with potential for future applications in neural prostheses and cell therapies.

Usando protocolos 1, 2 e 3 descritos aqui, um efeito estimulador sobre a diferenciação foi observada em células NG108-15 neuronais cultivadas com Au PN (Au NRs, poli (estirenossulfonato) -Revestido Au NRs e revestido de silica Au NRs) após o laser exposições entre 1,25 e 7,5 W · cm -2. Imagens confocal de rhodamineB marcado Au NRs demonstrado que as partículas foram internalizadas desde o dia 1 de incubação de 12. A localização foi predominantemente observada no citoplasma da célula, i...

Watch this Scientific Journal Video about Gold Nanorod-assisted Optical Stimulation of Neuronal Cells at JoVE.com

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

Ouro nanorod assistida estimulação óptica de Neuronal Cells

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared ...

gold-nanorod-assisted-optical-stimulation-of-neuronal-cells

Research

JoVE Journal

Neuroscience

8.9K visualizações.  Swinburne University of Technology. This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

Vídeo: Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

Using Affordable LED Arrays for Photo-Stimulation of Neurons

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single cells in response to electrical or pharmacological manipulations1,2. With the invention of methods to optically control genetically targeted neurons (optogenetics), researchers now have an unprecedented level of control over specific groups of neurons in the standard slice preparation. In particular, photosensitive channelrhodopsin-2 (ChR2) allows researchers to activate neurons with light3,4. By combining careful calibration of LED-based photostimulation of ChR2 with standard slice electrophysiology, we are able to probe with greater detail the role of adult-born interneurons in the olfactory bulb, the first central relay of the olfactory system. Using viral expression of ChR2-YFP specifically in adult-born neurons, we can selectively control young adult-born neurons in a milieu of older and mature neurons. Our optical control uses a simple and inexpensive LED system, and we show how this system can be calibrated to understand how much light is needed to evoke spiking activity in single neurons. Hence, brief flashes of blue light can remotely control the firing pattern of ChR2-transduced newborn cells.

Adult-born neurons expressing ChR2 can be manipulated in slice electrophysiological preparations in order to examine their contribution towards the function of olfactory neural circuits.

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 ...

Neurociência

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

Transdução Viral seletiva de Adultos-nascido neurônios olfativos de crônica In vivo Estímulo Optogenetic

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

Implantação de fibra óptica para Estimulação Crônica optogenética do Tecido Cerebral

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

Gold Nanoparticle Modified Carbon Fiber Microelectrodes for Enhanced Neurochemical Detection

For over 30 years, carbon-fiber microelectrodes (CFMEs) have been the standard for neurotransmitter detection. Generally, carbon fibers are aspirated into glass capillaries, pulled to a fine taper, and then sealed using an epoxy to create electrode materials that are used for fast scan cyclic voltammetry testing. The use of bare CFMEs has several limitations, though. First and foremost, the carbon fiber contains mostly basal plane carbon, which has a relatively low surface area and yields lower sensitivities than other nanomaterials. Furthermore, the graphitic carbon is limited by its temporal resolution, and its relatively low conductivity. Lastly, neurochemicals and macromolecules have been known to foul at the surface of carbon electrodes where they form non-conductive polymers that block further neurotransmitter adsorption. For this study, we modify CFMEs with gold nanoparticles to enhance neurochemical testing with fast scan cyclic voltammetry. Au3+ was electrodeposited or dipcoated from a colloidal solution onto the surface of CFMEs. Since gold is a stable and relatively inert metal, it is an ideal electrode material for analytical measurements of neurochemicals. Gold nanoparticle modified (AuNP-CFMEs) had a stability to dopamine response for over 4 h. Moreover, AuNP-CFMEs exhibit an increased sensitivity (higher peak oxidative current of the cyclic voltammograms) and faster electron transfer kinetics (lower &#916;EP or peak separation) than bare unmodified CFMEs. The development of AuNP-CFMEs provides the creation of novel electrochemical sensors for detecting fast changes in dopamine concentration and other neurochemicals at lower limits of detection. This work has vast applications for the enhancement of neurochemical measurements. The generation of gold nanoparticle modified CFMEs will be vitally important for the development of novel electrode sensors to detect neurotransmitters in vivo in rodent and other models to study neurochemical effects of drug abuse, depression, stroke, ischemia, and other behavioral and disease states.

In this study, we modify carbon-fiber microelectrodes with gold nanoparticles to enhance the sensitivity of neurotransmitter detection.

Nanopartícula de ouro modificado microeletrodos de fibra de carbono para detecção neuroquímica reforçada

For over 30 years, carbon-fiber microelectrodes (CFMEs) have been the standard for neurotransmitter detection. Generally, carbon fibers are aspirated into ...

Química

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

Preparação e Fotoacústica Análise de Veículos celulares contendo ouro nanobastões

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Bioengenharia

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

Controle óptico de células vivas atividade elétrica por conjugados Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

In vivo Optogenetic estimulação do sistema nervoso central de roedores

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

A estimulação optogenética do nervo auditivo

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.

Nanofios de Silício e Estimulação Óptica para Investigações de Acoplamento Elétrico Intra e Intercelular

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These ...

Optrode Array for Simultaneous Optogenetic Modulation and Electrical Neural Recording

During the last decade, optogenetics has become an essential tool for the investigation of neural signaling due to its unique capability of selective neural modulation or monitoring. As specific types of neuronal cells can be genetically modified to express opsin proteins, optogenetics enables optical stimulation or inhibition of the selected neurons. There have been several technological advances in the optical system for optogenetics. Recently, it was proposed to combine the optical waveguide for light delivery with electrophysiological recording to simultaneously monitor the neural responses to optogenetic stimulation or inhibition. In this study, an implantable optrode array (2x2 optical fibers) was developed with embedded multichannel electrodes. 
A light-emitting diode (LED) was employed as a light source, and a microfabricated microlens array was integrated to provide sufficient light power at the tip of the optical fibers. The optrode array system comprises the disposable part and the reusable part. The disposable part has optical fibers and electrodes, while the reusable part has the LED and electronic circuitry for light control and neural signal processing. The novel design of the implantable optrode array system is introduced in the accompanying video in addition to the procedure of the optrode implantation surgery, optogenetic light stimulation, and the electrophysiological neural recording. The results of in vivo experiments successfully showed time-locked neural spikes evoked by the light stimuli from hippocampal excitatory neurons of mice.

Here, we present the fabrication method of an optrode system with optical fibers for light delivery and an electrode array for neural recording. In vivo experiments with transgenic mice expressing channelrhodopsin-2 show the feasibility of the system for simultaneous optogenetic stimulation and electrophysiological recording.

Oprode Array para modulação optogenética simultânea e gravação neural elétrica

During the last decade, optogenetics has become an essential tool for the investigation of neural signaling due to its unique capability of selective ...