O trabalho foi financiado pelo Centro de NIH / Nacional para Medicina Complementar e Alternativa (R21AT00978) (1-P01-002048-01) (K01-AT-002166-01), o Centro Nacional de Pesquisa de Recursos (P41RR14075), a Doença Mental e Neuroscience Discovery Institute (MIND) eo Cérebro Projeto Grant (NS 34.189).

Parte 1: preparação consentimento e sujeita Informado Voluntários adultos saudáveis ​​são recrutados para o estudo de acordo com o Conselho de Revisão Institucional aprovado procedimentos. Consentimento informado pelo sujeito é assinado antes de iniciar o estudo. Parte 2: Configurar o equipamento de monitorização fisiológica A AD Instruments sistema PowerLab permite que os dados fisiológicos para ser sincronizado com a varredura fMRI usando o sinal de disparo fMRI gravados em um canal de entrada. Este é um sistema baseado em laptop com entradas para ECG, respiração, resposta de condutância da pele, oximetria de pulso, e qualquer outro sinal fisiológico que precisam ser registradas. <ol><li> Se um sistema de correias respiratória está sendo usado, o transdutor deve ser conectado a uma tomada elétrica na sala ao lado do scanner, com o tubo que conduz a correia alimentada via de entrada para a sala de digitalização e um conector BNC levando à PowerLab na scan sala de controle. </li><li> Se um gatilho está sendo usado para sincronizar os dados fisiológicos com os dados fMRI, em seguida, um cabo BNC também deve ligar o sinal de disparo para o scanner de ressonância magnética PowerLab na sala de controle de verificação. </li><li> Reúna dispositivos de monitoramento. Quatro eletrodos ECG serão necessários, bem como um cabo de ECG conectado a uma MR-compatível In Vivo rollcart sistema. </li><li> Se condutância da pele está sendo medido, ligue os conectores condutância da pele na caixa de breakout na sala de digitalização e tem gel pele condutância pronto na sala de digitalização. </li></ol> Parte 3: Assunto Preparando-se para MR digitalização <ol><li> É importante para afastar a ansiedade excessiva e fadiga no dia do exame, porque estado básico podem afetar a resposta do cérebro a estímulos de acupuntura. Sugerir o assunto comer uma refeição leve antes do procedimento para que as reações fraqueza e indesejáveis ​​podem ser evitados. </li><li> Instruir a pessoa para se deitar em decúbito dorsal na máquina usando tampões de ouvido para suprimir o ruído scanner e com a cabeça imobilizada por suportes almofadados. Uma vez que a relação sinal / ruído de exames estimulação da acupuntura é baixo quando comparado com o motor comum ou tarefas visuais, os dados tende a ser mais facilmente corrompidos pelo movimento da cabeça. Um suporte é colocado sob os joelhos para manter os calcanhares formam a superfície da maca para reduzir o movimento do corpo. A bobina de cabeça é presa e almofadas são ajustados para o conforto. Para uma sessão média de varredura de 2 horas, o conforto é vital para a digitalização sem interrupções. </li><li> Se o monitoramento fisiológico é desejada, assunto deve ser preparado de acordo com a seção anterior, antes de ser colocado no scanner. É desejável para monitorar o estado de vigília do sujeito com rastreamento ocular durante os exames, se disponível. </li><li> Montar 4 eletrodos ECG de peito esquerdo do sujeito superior (para o sexo feminino, tomar as precauções de privacidade tais como a redução da blinds na janela para a baía na sala de controle de verificação e fechando a porta scanner). Conecte o cabo aos eletrodos e ao Vivo No dispositivo. Por razões de segurança este cabo e todos os outros cabos levando ao assunto deve ser executado diretamente ao longo da varredura da tabela, sem formar qualquer loops. Ligue o dispositivo No Vivo e verificar a qualidade do sinal ECG. Ajustar posicionamento dos eletrodos se necessário. </li><li> Preencha os poços dos eletrodos SCR com gel condutor. Este gel SCR especial deve ser isotônica à pele. Executar aberta de zero no circuito de software gráfico. Anexar eletrodos SCR para 2 adjacentes as pontas dos dedos distal da mão do sujeito por envolver o velcro em torno de tão firmemente como é confortável. </li></ol> Parte 4: Funcional seqüências de ressonância magnética de imagens sob medida para a estimulação de acupuntura. <ol><li> Usamos um 1.5 ou 3.0 Tesla Siemens Trio sistema MRI (Siemens, Erlangen, Alemanha) equipado para echo planar imaging com uma bobina de cabeça padrão quadrático. </li><li> Nossos estudos ao longo dos anos, consistentemente, demonstrado que a acupuntura teve ação extensiva em relação aos sistemas funcionais em múltiplos níveis do cérebro. As estruturas localizadas na base do cérebro como a amígdala, o hipocampo, córtex pré-frontal medial ventral e áreas subgenual são particularmente suscetíveis a artefatos de susceptibilidade. É importante verificar cada EPI scan funcional para afastar a perda do sinal nestas regiões, encontramos diferenças na perda de sinais entre dois modelos de um scanner Siemens com força campo semelhante e com uma seqüência semelhante de ressonância magnética para a coleta de dados. </li><li> Fatias mais finas do cérebro são preferíveis para reduzir os artefatos de susceptibilidade. Fatias de 3 mm de espessura com 0,6 mm ou 0,75 mm de gap a orientação sagital ou axial são usados ​​para cobrir todo o cérebro, incluindo o tronco encefálico e cerebelo, dependendo da capacidade de memória do sistema de imagem. Outras orientações e espessura de corte pode ser usado para imagiologia cerebral parcial. A atenção especial que pagamos para estes problemas contribuem para o maisachados consistentes de efeitos da acupuntura sobre as estruturas na base do cérebro. </li><li> Os exames funcionais são adquiridos com um T2 *- seqüência gradiente echo ponderada (TR / TE 4000/30 ms, FOV de 200 mm, ângulo de matriz, 64x64 virar 90 °, uma fatia de espessura de 3,0 mm, abertura 0,6 mm). Adquirimos um total de 150 pontos no tempo em 10 min. Coleção de imagens é precedido por quatro varreduras dummy para permitir equilíbrio do sinal de fMRI. O TE mais curto reduz artefatos de susceptibilidade. A TR relativamente longo permite a cobertura do cérebro inteiro com alta resolução espacial. </li></ol> Parte 5: O localizador inicial e scans anatômica <ol><li> Antes de varreduras funcionais com a acupuntura são executados, uma varredura localizador, uma varredura de alta resolução anatômica, e os T1 ponderada 3D MP-RAGE são adquiridos para garantir o posicionamento correto e solidez anatômica. Observação geral neste momento pode garantir que scans desnecessários não são executados. O localizador, uma varredura muito curto, garante que todo o cérebro está incluído na digitalização e permite alinhá-lo com a linha AC-PC. Se houver qualquer suspeita de anomalias, os pesquisadores vão enviar imagens para um radiologista treinado, mas não devem fazer quaisquer declarações sobre o que está no scan a indivíduos. </li><li> Os exames iniciais de aproximadamente 20 minutos total. Visualização breve pode dizer o investigador se ele vai ser útil para continuar a digitalização. Este é um bom momento para falar com o assunto e se certificar de que eles ainda são confortáveis ​​e não tendo qualquer tipo de efeitos adversos de estar no ímã. </li></ol> Parte 6: Acupuntura com o monitoramento fMRI Das duas formas de tratamento (manipulação de agulha manual e estimulação elétrica), este vídeo será limitada a acupuntura manual, que tem uma história muito mais tempo de uso clínico. Estimulação da acupuntura é administrada em corridas duplicado para acupoints clássica LI4 na mão, LV3 no pé ou na perna ST36, empregando estéril, um tempo de usar apenas agulhas de acupuntura. <ol><li> Instruir indivíduos para relaxar, manter os olhos fechados e abster-se de movimentos durante a digitalização. Para evitar a estimulação nociva, que especificamente instruir o assunto para levantar um dedo, se qualquer uma das sensações da acupuntura se aproxima de uma pontuação de 8, e levantar dois dedos em caso de dor aguda de qualquer grau. </li><li> Depois de inserir a agulha pré-teste, a sensibilidade do sujeito e da tolerância para a manipulação de agulhas para ajudar a escolher a profundidade de inserção ea força de manipulação para coleta de dados. </li><li> Durante os 10 minutos de imagem funcional, a agulha é girada por 2 min durante M1 e M2, e deixado no local durante os períodos de descanso R1 (2min)), R2 (3min), R3 (1 min). O acupunturista garante que não se aproximar ou deixar o assunto imediatamente antes ou após o período de estimulação, como até mesmo movimento que poderiam afetar os resultados. (Paradigm, Fig. 1) <img src="/files/ftp_upload/1190/1190fig1.n.jpg" alt="Figura 1" /> Figura 1. Acupuntura Paradigm fMRI. </li><li> Após a conclusão da estimulação de acupuntura, o acupunturista registra a força com que a agulha resiste manipulação. No final de cada varredura, outro investigador pessoal pede ao sujeito que grau as sensações experimentadas em uma escala de 1-10. A lista, dor, dor, pressão, plenitude, peso, dormência, formigamento, dor surda, dor aguda, é baseado na experiência da comunidade acupunturista e anteriores dados experimentais ao invés de questionários dor na literatura. A resposta psicofísica é classificado como um grupo deqi típicas e um outro grupo de deqi misturado com dor aguda com base nesses registros. </li><li> Fale com os assuntos entre as varreduras para ter certeza de que eles se sintam confortáveis ​​e que ficar acordado. Se qualquer parte de seu corpo começa a cãibra ou se sentir desconfortável, a equipe de pesquisa no local devem tomar medidas adequadas para aliviar o desconforto. Movimentos gerados pelo sujeito causaria corrupção de dados. Assuntos ocasionais pode adormecer durante a digitalização, o que afetaria a resposta hemodinâmica. </li></ol> Parte 7: Processamento de Dados <ol><li> Múltiplos níveis de análise pode ser executado. Um modelo linear geral usa os períodos de descanso como uma base para determinar a mudança de sinal BOLD% MR em cada voxel do cérebro durante a tarefa. Também determina a significância estatística dessas mudanças e remove os dados BOLD que não alcançam 3 voxels contíguos em um determinado valor-p. </li><li> Executar semente baseada em análise de correlação cruzada para mostrar a conectividade funcional entre uma região neurológicas e do resto do cérebro. Isto é feito selecionando uma das regiões de referência e comparando o seu tempo claro com que de cada voxel outras no cérebro. Outras análises, tais como análise de componentes independentes, análise de agrupamento fuzzy, modelagem de equações estruturais, Causalidade Granger pode ser aplicada. </li><li> Dados fisiológicos podemser analisada pela média de resposta dentro de janelas relacionadas com acupuntura estímulo da agulha. Por exemplo, dados de ECG podem ser anotadas para QRS tempo complexos seguida da análise da freqüência cardíaca e variabilidade da freqüência cardíaca. </li></ol> Parte 8: Resultados <img src="/files/ftp_upload/1190/1190fig2.n.jpg" alt="Figura 2" /> Figura 2. Resposta hemodinâmica em acupuntura deqi versus estimulação tátil à direita LI4, ST36 e LV3 (52 runs/37 indivíduos). Os agrupamentos de estruturas desactivadas no (córtex pré-frontal medial) MPFC (1), MPC (córtex parietal medial) (2), MTL (lobo temporal medial) (3), foram mais proeminentes em acupuntura (Fig A) do que em estimulação tátil (Fig B). Desativação extensa do BA22/21 direito (4) em acupuntura não foi observado em estimulação tátil. A BA43 sensório-motor (5) e córtex de associação (6) demonstraram mais ativação na estimulação tátil do que em acupuntura (não mostrados). Curiosamente, a ínsula anterior direita (7), foi ativado em acupuntura e não na estimulação tátil. (P &lt;0,0001). <img src="/files/ftp_upload/1190/1190fig3.n.jpg" alt="Figura 3" /> Figura 3. Hemodinâmicos em resposta deqi (52 runs/37 assuntos) versus deqi misturado com dor aguda (52 runs/29 indivíduos) durante a estimulação de acupuntura à direita LI4, ST36 e LV3, A desativação proeminente do MPFC (1), MPC ( 2) e MTL (3) visto com deqi dor ausente (Fig. A) foi atenuada na presença de dor (Fig B). Com dor, a ativação do córtex sensório-motor e de associação (4) tornou-se mais proeminente, e um subconjunto das regiões límbicas e paralímbica como o MIDC / SMA (5), postC_BA23 (6), Amy (7), e vermis cerebelar ( 8) tornou-se ativado. O direito anterior ínsula (9) foi marcada ativado em deqi enquanto que as áreas menores de ínsula anterior e posterior (10) foram ativados bilateralmente na dor. P &lt;0,0001. <img src="/files/ftp_upload/1190/1190fig4.n.jpg" alt="Figura 4" /> Figura 4 Sobreposição de límbico-paralímbica-neocortical rede (LPNN) em acupuntura com a rede de modo default (DMN);. A resposta BOLD da LPNN por GLM análise (Fig.A) e pelo modelo livre de análise de Cluster Fuzzy (Fig. B ) durante a estimulação de acupuntura em LI4, ST36 e LV3 mostram padrões praticamente idênticos com o DMN (Fig.C, adaptado de Shulman et al, 1997). Grupos de regiões com sinal diminui coerente aparecem no córtex pré-frontal medial (1), póstero-medial córtex (2) e do lobo temporal (3). p &lt;0,0001.

<ol>
	<li>Hui, K. K. S., Liu, J., Marina, O., Napadow, V., Haselgrove, C., Kwong, K. K., Kennedy, D. N., Makris, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+integrated+response+of+the+human+cerebro-cerebellar+and+limbic+systems+to+acupuncture+stimulation+at+ST+36+as+evidenced+by+fMRI.">The integrated response of the human cerebro-cerebellar and limbic systems to acupuncture stimulation at ST 36 as evidenced by fMRI.</a> Neuroimage. 27 (3), 479-496 (2005).</li><li>Hui, K. K. S., Marina, O., Claunch, J. D., Nixon, E. E., Fang, J., Liu, J., Li, M., Napadow, V., Vangel, M., Makris, N., Chan, S. T., Kwong, K. K., Rosen, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acupuncture+mobilizes+the+brain's+default+mode+and+its+anti-correlated+network+in+healthy+subjects.">Acupuncture mobilizes the brain's default mode and its anti-correlated network in healthy subjects.</a> Brain Res. 1287, 84-103 (2009).</li><li>Napadow, V., Dhond, R. P., Purdon, P., Kettner, N., Makris, N., Kwong, K. K., Hui, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlating+acupuncture+fMRI+in+the+human+brainstem+with+heart+rate+variability.">Correlating acupuncture fMRI in the human brainstem with heart rate variability.</a> Conf Proc IEEE Eng Med Biol Soc. 5, 4496-4499 (2005).</li><li>Shulman, G. L., Corbetta, M., Buckner, R. L., Raichle, M. E., Fiez, J. A., Miezin, F. M., Petersen, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Top-down+modulation+of+early+sensory+cortex.">Top-down modulation of early sensory cortex.</a> Cereb. Cortex. 7 (3), 193-206 (1997).</li></ol>

O emprego de técnicas adequadas, a experiência do acupunturista e um tamanho de amostra grande para permitir a separação de efeitos de confusão de estímulos nocivos inadvertida são importantes na aquisição e interpretação de dados de fMRI acupuntura. Nossos estudos de vários pontos de acupuntura clássicos indicam que a acupuntura modula a rede límbico, um sistema regulador importante intrínseca do cérebro humano. A rede mostra sobreposição significativa com o modo padrão do cérebro em repouso. Estas ...

monitoring acupuncture effects on human brain by fmri

Ressonância magnética funcional é usado para estudar os efeitos da acupuntura sobre a resposta BOLD ea conectividade funcional do cérebro humano. Resultados demonstram que a acupuntura mobiliza uma rede límbico-paralímbica-neocortical e seu anti-correlacionados rede sensório / paralímbica em múltiplos níveis do cérebro e que a resposta hemodinâmica é influenciada pela resposta psicofísica. Monitoração fisiológica podem ser realizados para explorar a resposta periférica da função do nervo autonômico. Este vídeo descreve os estudos realizados em LI4 (Hegu), ST36 (Zusanli) e LV3 (Taichong), pontos de acupuntura clássicos que são comumente usados ​​para ações modulatórios e de redução de dor. Algumas questões que requerem atenção nas aplicações de fMRI para a investigação de acupuntura são anotados.

Watch this Scientific Journal Video about Monitoring Acupuncture Effects on Human Brain by fMRI at JoVE.com

Monitoring Acupuncture Effects on Human Brain by fMRI

Monitoramento FMRI e fisiológicos é usado para estudar os efeitos da acupuntura sobre os sistemas nervoso central e periférico. Acupuntura mobiliza uma rede límbico-paralímbica-neocortical, com grande sobreposição com a rede de modo padrão, para modular a atividade neurológica, possivelmente relacionado ao seu efeito autônomo no sistema nervoso periférico.

Monitoramento Effects Acupuntura em Cérebro Humano por fMRI

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate ...

monitoring-acupuncture-effects-on-human-brain-by-fmri

Research

JoVE Journal

Biology

15.2K visualizações.  Massachusetts General Hospital and Harvard Medical School. Monitoramento FMRI e fisiológicos é usado para estudar os efeitos da acupuntura sobre os sistemas nervoso central e periférico. Acupuntura mobiliza uma rede límbico-paralímbica-neocortical, com grande sobreposição com a rede de modo padrão, para modular a atividade neurológica, possivelmente relacionado ao seu efeito autônomo no sistema nervoso periférico.

Vídeo: Monitoring Acupuncture Effects on Human Brain by fMRI

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Estudos genéticos de proteínas de reparo do DNA Humano Utilizando Levedura como um sistema modelo

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Biologia

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Analisando a função de GTPases Pequenas pela microinjeção de Plasmids em Polarized Células Epiteliais

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

Mapear as sequelas de Theta Explosão Estimulação no córtex auditivo humano com Imagem Funcional

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

Exame<em&gt; Drosophila</em&gt; Larval células terminais traqueais por microscopia de luz

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Método de Avaliação de efeitos de uma gama de comprimentos de onda e intensidades de Terapia da Luz Vermelha / near-infrared sobre o estresse oxidativo<em&gt; In Vitro</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees collect and convert to beebread by placing colonies in a specially constructed enclosed flight area. Methods were developed to analyze the protein and amino acid composition of the pollen and beebread. We also describe how consumption of the beebread was measured and methods used to determine adult worker bee hemolymph protein titers after feeding on beebread for 4, 7 and 11 days after emergence. Methods were applied to determine if genotype affects the conversion of pollen to beebread and the rate that bees consume and acquire protein from it. Two subspecies (European and Africanized honey bees; EHB and AHB respectively) were provided with the same pollen source. Based on the developed methods, beebread made by both subspecies had lower protein concentrations and pH values than the pollen. In general, amino acid concentrations in beebread made by either EHB or AHB were similar and occurred at higher levels in beebread than in pollen. Both AHB and EHB consumed significantly more of the beebread made by AHB than by EHB. Though EHB and AHB consumed similar amounts of each type of beebread, hemolymph protein concentrations in AHB were higher than in EHB. Differences in protein acquisition between AHB and EHB might reflect environmental adaptations related to the geographic region where each subspecies evolved. These differences could contribute to the successful establishment of AHB populations in the New World because of the effects on brood rearing and colony growth.

Here are methods to quantify nutrients in pollen before and after its conversion to beebread by two subspecies of honeybees. We describe techniques to measure beebread consumption and resulting protein titers in both subspecies.

Métodos para comparar Nutrientes na beebread Feito por africanizadas e mel europeu abelhas e os efeitos sobre Títulos Hemolinfa Proteína

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

A quantificação da Adesividade celular endotelial<em&gt; In Vitro</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

Monitoramento retículo endoplasmático homeostase do cálcio Usando um<em&gt; Gaussia</em&gt; Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

O isolamento de regiões específicas genômico e identificação de moléculas pela Associated enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecal Análise de glicocorticóides: Non-invasive Monitoring Adrenal em eqüídeos

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...