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>

<ol>
	<li>Richter, C. P., Matic, A. I., Wells, J. D., Jansen, E. D., Walsh, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stimulation+with+optical+radiation.">Neural stimulation with optical radiation.</a> Laser. Photonics Rev. 5 (1), 68-80 (2011).</li><li>Dittami, G. M., Rajguru, S. M., Lasher, R. A., Hitchcock, R. W., Rabbitt, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+calcium+transients+evoked+by+pulsed+infrared+radiation+in+neonatal+cardiomyocytes.">Intracellular calcium transients evoked by pulsed infrared radiation in neonatal cardiomyocytes.</a> J. Physiol. 589 (6), 1295-1306 (2011).</li><li>Thompson, A. C., Wade, S. A., Brown, W. G. A., Stoddart, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+of+light+absorption+in+tissue+during+infrared+neural+stimulation.">Modeling of light absorption in tissue during infrared neural stimulation.</a> J. Biomed. Opt. 17 (7), 075002-075002 (2012).</li><li>Thompson, A. C., Wade, S. A., Cadusch, P. J., Brown, W. G., Stoddart, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+of+the+temporal+effects+of+heating+during+infrared+neural+stimulation.">Modeling of the temporal effects of heating during infrared neural stimulation.</a> J. Biomed. Opt. 18 (3), 035004 (2013).</li><li>Huang, H., Delikanli, S., Zeng, H., Ferkey, D. M., Pralle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+control+of+ion+channels+and+neurons+through+magnetic-field+heating+of+nanoparticles.">Remote control of ion channels and neurons through magnetic-field heating of nanoparticles.</a> Nat. Nanotechnol. 5 (8), 602-606 (2010).</li><li>Farah, N., 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=Holographically+patterned+activation+using+photo-absorber+induced+neural-thermal+stimulation.">Holographically patterned activation using photo-absorber induced neural-thermal stimulation.</a> J. Neural. Eng. 10 (5), (2013).</li><li>Ciofani, G., 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=Enhancement+of+neurite+outgrowth+in+neuronal-like+cells+following+boron+nitride+nanotube-mediated+stimulation.">Enhancement of neurite outgrowth in neuronal-like cells following boron nitride nanotube-mediated stimulation.</a> ACS Nano. 4 (10), 6267-6277 (2010).</li><li>Kim, J. 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=Enhancement+of+neurite+outgrowth+in+PC12+cells+by+iron+oxide+nanoparticles.">Enhancement of neurite outgrowth in PC12 cells by iron oxide nanoparticles.</a> Biomaterials. 32 (11), 2871-2877 (2011).</li><li>Myroshnychenko, V., 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=Modelling+the+optical+response+of+gold+nanoparticles.">Modelling the optical response of gold nanoparticles.</a> Chem. Soc. Rev. 37 (9), 1792-1805 (2008).</li><li>Choi, W. I., Sahu, A., Kim, Y. H., Tae, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photothermal+cancer+therapy+and+imaging+based+on+gold+nanorods.">Photothermal cancer therapy and imaging based on gold nanorods.</a> Ann. Biomed. Eng. 40 (2), 534-546 (2011).</li><li>Zhan, Q., Qian, J., Li, X., He, 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+study+of+mesoporous+silica-encapsulated+gold+nanorods+as+enhanced+light+scattering+probes+for+cancer+cell+imaging.">A study of mesoporous silica-encapsulated gold nanorods as enhanced light scattering probes for cancer cell imaging.</a> Nanotechnology. 21 (5), 055704 (2010).</li><li>Paviolo, C., 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=Laser+exposure+of+gold+nanorods+can+increase+neuronal+cell+outgrowth.">Laser exposure of gold nanorods can increase neuronal cell outgrowth.</a> Biotechnol. Bioeng. 110 (8), 2277-2291 (2013).</li><li>Paviolo, C., Haycock, J. W., Cadusch, P. J., McArthur, S. L., Stoddart, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+exposure+of+gold+nanorods+can+induce+intracellular+calcium+transients.">Laser exposure of gold nanorods can induce intracellular calcium transients.</a> J. Biophotonics. 7 (10), 761-765 (2014).</li><li>Yong, J., 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=Gold-nanorod-assisted+near-infrared+stimulation+of+primary+auditory+neurons.">Gold-nanorod-assisted near-infrared stimulation of primary auditory neurons.</a> Adv. Healthcare Mater. , (2014).</li><li>Eom, K., 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=Enhanced+infrared+neural+stimulation+using+localized+surface+plasmon+resonance+of+gold+nanorods.">Enhanced infrared neural stimulation using localized surface plasmon resonance of gold nanorods.</a> Small. , (2014).</li><li>Juste, J., Pastoriza-Santos, I., Liz-Marz&#225;n, L. M., 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> Coordination Chemistry Reviews. (17-18), 1870-1901 (2005).</li><li>Shang, J., Gao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+counting:+towards+accurate+determination+of+the+molar+concentration.">Nanoparticle counting: towards accurate determination of the molar concentration.</a> Chem. Soc. Rev. 43 (21), 7267-7278 (2014).</li><li>Sharma, V., Park, K., Srinivasarao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape+separation+of+gold+nanorods+using+centrifugation.">Shape separation of gold nanorods using centrifugation.</a> Proc. Natl. Acad. Sci. 106 (13), 4981-4985 (2009).</li><li>Kaewkhaw, R., Scutt, A. M., Haycock, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+site+influences+the+differentiation+of+adipose-derived+stem+cells+for+schwann-cell+phenotype+and+function.">Anatomical site influences the differentiation of adipose-derived stem cells for schwann-cell phenotype and function.</a> Glia. 59 (5), 734-749 (2011).</li><li>Brown, W. G. A., Needham, K., Nayagam, B. A., Stoddart, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+cell+patch+clamp+for+investigating+the+mechanisms+of+infrared+neural+stimulation.">Whole cell patch clamp for investigating the mechanisms of infrared neural stimulation.</a> JoVE. (77), (2013).</li><li>Cadusch, P. J., Hlaing, M. M., Wade, S. A., McArthur, S. L., Stoddart, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methods+for+fluorescence+background+subtraction+from+Raman+spectra.">Improved methods for fluorescence background subtraction from Raman spectra.</a> J. Raman Spectrosc. 44 (11), 1587-1595 (2013).</li><li>Daud, M. F. B., Pawar, K. C., Claeyssens, F., Ryan, A. J., Haycock, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+aligned+3D+neuronal-glial+co-culture+model+for+peripheral+nerve+studies.">An aligned 3D neuronal-glial co-culture model for peripheral nerve studies.</a> Biomaterials. 33 (25), 5901-5913 (2012).</li><li>Jung, 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=Intracellular+gold+nanoparticles+increase+neuronal+excitability+and+aggravate+seizure+activity+in+the+mouse+brain.">Intracellular gold nanoparticles increase neuronal excitability and aggravate seizure activity in the mouse brain.</a> PLoS ONE. 9 (3), e91360 (2014).</li><li>Salinas, K., Kereselidze, Z., DeLuna, F., Peralta, X., Santamaria, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+extracellular+application+of+gold+nanostars+increases+hippocampal+neuronal+activity.">Transient extracellular application of gold nanostars increases hippocampal neuronal activity.</a> J. Nanobiotechnology. 12 (1), 31 (2014).</li><li>Ebbesen, C. L., Bruus, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+laser-induced+heating+in+optical+neuronal+guidance.">Analysis of laser-induced heating in optical neuronal guidance.</a> J. Neurosci. Meth. 209 (1), 168-177 (2012).</li><li>Iwanaga, 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=Location-dependent+photogeneration+of+calcium+waves+in+HeLa+cells.">Location-dependent photogeneration of calcium waves in HeLa cells.</a> Cell Biochem. Biophys. 45 (2), 167-176 (2006).</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=Determination+of+the+minimum+temperature+required+for+selective+photothermal+destruction+of+cancer+cells+with+the+use+of+immunotargeted+gold+nanoparticles.">Determination of the minimum temperature required for selective photothermal destruction of cancer cells with the use of immunotargeted gold nanoparticles.</a> Photochem. Photobiol. 82 (2), 412-417 (2006).</li><li>Connor, E. E., Mwamuka, J., Gole, A., Murphy, C. J., Wyatt, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanoparticles+are+taken+up+by+human+cells+but+do+not+cause+acute+cytotoxicity.">Gold nanoparticles are taken up by human cells but do not cause acute cytotoxicity.</a> Small. 1 (3), 325-327 (2005).</li><li>Isomaa, B., Reuter, J., Djupsund, B. 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 border="0" cellpadding="0" cellspacing="0">
	<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-&#946;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></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment name</td>
			<td>Company</td>
			<td>Catalogue Number</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>&#956;-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

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

Combinando Estimulação Magnética Transcraniana e fMRI para o Exame da rede de modo padrão

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

Neurociência

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 ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

O dispositivo NeuroStar TMS: Conduzindo a FDA Aprovado protocolo para o tratamento da depressão

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Em células vivas, imagens de células precursoras de órgãos sensoriais na Intact Drosophila As pupas

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Controlo óptico de uma proteína neuronal utilizando um ácido Unnatural Amino geneticamente codificados em neurónios

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Aumento Time-dependente na resposta da rede à estimulação de culturas de pilha neuronais em matrizes do micro-elétrodo

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Tomografia de coerência óptica: Imagens Mouse ganglionares da retina células In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification 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 ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

Diferenciação neuronal de células-tronco embrionárias do rato in vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

GM-Free Generation of Blood-Derived Neuronal Cells

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

Geração livre de células neuronais derivadas do sangue

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...