El trabajo fue apoyado por el Centro de NIH / Nacional de Medicina Complementaria y Alternativa (R21AT00978) (1-P01-002048-01) (K01-A-002166 a 01), el Centro Nacional para Recursos de Investigación (P41RR14075), la enfermedad mental y Neurociencia del Instituto Discovery (MIND) y el cerebro del proyecto de subvención (NS 34189).

Parte 1: preparación de consentimiento informado y el tema Voluntarios sanos adultos son reclutados para el estudio de acuerdo con la Junta de Revisión Institucional de los procedimientos aprobados. Consentimiento informado por parte del sujeto se firma antes de comenzar el estudio. Parte 2: Configuración del equipo de monitorización fisiológica Al instrumento del sistema PowerLab permite que los datos fisiológicos para sincronizarse con la exploración de resonancia magnética funcional mediante el uso de la señal de disparo fMRI se registran en un canal de entrada. Este es un sistema portátil basado en los insumos para ECG, respiración, la respuesta de conductancia de la piel, la oximetría de pulso, y cualquier otra señal fisiológica que necesitan ser registradas. <ol><li> Si un sistema de cinturón de las vías respiratorias se está utilizando, el transductor debe estar conectado a una toma de corriente en la habitación contigua a la del escáner, con el tubo que alimenta a la banda a través de la entrada en la sala de exploración y un conector BNC que lleva a la PowerLab en el exploración sala de control. </li><li> Si un disparador se utiliza para sincronizar los datos fisiológicos de los datos de la fMRI, y luego un cable BNC también debe conectar la señal de activación del escáner de resonancia magnética para el PowerLab en la sala de control de escaneo. </li><li> Se reúnen los dispositivos de vigilancia. Cuatro electrodos de ECG se necesita, así como un cable de ECG conectado a un MR-compatible En Vivo rollcart sistema. </li><li> Si conductancia de la piel que se está midiendo, conecte los conectores de conductancia de la piel en la caja de conexiones en la sala de exploración, y han de gel de conductancia de la piel preparada en la sala de exploración. </li></ol> Parte 3: Título Preparación para la RM <ol><li> Es importante descartar que la ansiedad excesiva y fatiga en el día de la exploración básica estatal, porque puede afectar la respuesta del cerebro a la estimulación de acupuntura. Sugerir el tema ingiera una comida ligera antes de la intervención para que las reacciones adversas y la debilidad puede ser evitado. </li><li> Instruir a los sujetos a decúbito supino en la máquina usar tapones para los oídos para suprimir el ruido del escáner y con la cabeza inmovilizada por soportes acolchados. Dado que la relación señal / ruido de las exploraciones de acupuntura estimulación es baja en comparación con un motor común o tareas visuales, los datos tienden a ser más fácilmente dañados por el movimiento de la cabeza. Un soporte se coloca debajo de las rodillas para mantener los talones forman la superficie de la camilla para reducir el movimiento del cuerpo. La bobina de la cabeza se fija y los cojines se ajustan para mayor comodidad. Para una sesión de media exploración de 2 horas, la comodidad es vital para el escaneado ininterrumpido. </li><li> Si se desea una monitorización fisiológica, tema debe ser preparado de acuerdo con el apartado anterior antes de ser puesto en el escáner. Es conveniente vigilar el estado de vigilia del sujeto con el eye-tracking durante el análisis si está disponible. </li><li> Adjuntar cuatro electrodos de ECG en el pecho de arriba a la izquierda del sujeto (por sujetos de sexo femenino, tome las precauciones de privacidad como la reducción de las persianas de la ventana de la bahía en la sala de control de escáner y cierre la puerta del escáner). Conecte el cable a los electrodos y el dispositivo de En Vivo. Para la seguridad de este cable y todos los otros cables que conducen a la materia debe correr directamente a lo largo de la mesa de exploración, sin formar bucles. Encienda el dispositivo En Vivo y verificar la calidad de la señal del ECG. Ajuste la posición de electrodo si es necesario. </li><li> Llenar los pozos de los electrodos con gel conductor SCR. Este gel especial SCR debe ser isotónica con la piel. Realizar abierto cero del circuito en el software gráfico. Coloque los electrodos SCR a 2 pastillas al lado distal del dedo de la mano del sujeto, envolviendo el velcro alrededor de lo más fuerte que se sienta cómodo. </li></ol> Parte 4: RMN funcional secuencias de imágenes a medida para obtener imágenes de la estimulación de acupuntura. <ol><li> Utilizamos un 1,5 o 3,0 Tesla MRI Siemens Trio sistema (Siemens, Erlangen, Alemania) equipado de echo imágenes planas con una bobina de la cabeza cuadrática estándar. </li><li> Nuestros estudios en los últimos años siempre ha demostrado que la acupuntura había una amplia acción sobre los sistemas funcionales en múltiples niveles del cerebro. Las estructuras localizadas en la base del cerebro como la amígdala, el hipocampo, la corteza prefrontal ventral medial y áreas subgenual son particularmente susceptibles a artefactos de susceptibilidad. Es importante revisar cada exploración funcional del PAI para descartar la pérdida de señal en estas regiones, se encontraron diferencias en la pérdida de las señales entre dos modelos de un escáner de Siemens con la intensidad de campo similar y con una secuencia similar de resonancia magnética para la recolección de datos. </li><li> Cortes más delgados del cerebro son preferibles para la reducción de los artefactos de susceptibilidad. Rebanadas de 3 mm de espesor, con 0,6 mm o 0,75 mm de distancia en la orientación sagital o axial se utilizan para cubrir todo el cerebro incluido el tallo cerebral y el cerebelo en función de la capacidad de memoria del sistema de imagen. Otras orientaciones y grosor de corte se puede utilizar para obtener imágenes del cerebro parcial. La especial atención que prestamos a estas cuestiones contribuyen a la mayorhallazgos consistentes de los efectos de la acupuntura en las estructuras en la base del cerebro. </li><li> Las exploraciones funcionales se adquieren con una secuencia de T2 *- ponderado de eco de gradiente (TR / TE 4000/30 ms, FOV 200 mm, matriz de 64x64 ángulo, giro 90 º, grosor de corte de 3,0 mm, 0,6 mm de distancia). Hemos adquirido un total de 150 puntos de tiempo en 10 minutos. Colección de imágenes es precedida por cuatro exploraciones ficticias para permitir el equilibrio de la señal fMRI. Cuanto más corto sea TE reduce artefactos de susceptibilidad. La TR permite una cobertura relativamente largo de todo el cerebro con una alta resolución espacial. </li></ol> Parte 5: El localizador inicial y exploraciones anatómicas <ol><li> Antes de las exploraciones funcionales con la acupuntura se ejecutan, una exploración de localizador, un escáner de alta resolución anatómica, y el T1 ponderado 3D RAGE MP-son adquiridas para asegurar el correcto posicionamiento y solidez anatómica. Observación general en este momento podemos asegurar que las exploraciones innecesarias, no se ejecutan. El localizador, un análisis muy corto, asegura que el cerebro está incluido en el análisis y le permite alinearse con la línea AC-PC. Si hay alguna anomalía sospechosa, los investigadores enviar imágenes a un radiólogo, pero no debe hacer ninguna afirmación sobre lo que está en la exploración de temas. </li><li> El análisis inicial de unos 20 minutos en total. Breve visión puede decir que el investigador si será útil para continuar la exploración. Este es un buen momento para hablar del tema y asegurarse de que todavía son cómodas y no están experimentando los efectos adversos de estar en el imán. </li></ol> Parte 6: La acupuntura con el monitoreo fMRI De las dos formas de tratamiento (manipulación de las agujas manual y la estimulación eléctrica), este vídeo se limitará a la acupuntura manual, que tiene una historia mucho más larga de uso clínico. Estimulación de acupuntura se administra en carreras duplicado a los puntos de acupuntura clásica LI4 en la mano, LV3 en el pie o ST36 en la pierna, el empleo de estériles, de un solo uso agujas de acupuntura. <ol><li> Instruya a los sujetos para relajarse, mantener los ojos cerrados y se abstengan de movimientos durante la exploración. Para evitar la estimulación nociva, específicamente instruir el tema de elevar un dedo si alguna de las sensaciones de la acupuntura se acerca a una puntuación de 8, y elevar dos dedos en caso de dolor agudo de cualquier grado. </li><li> Después de insertar la aguja, pre-test de sensibilidad del sujeto y la tolerancia a la aguja de la manipulación para ayudar a elegir la profundidad de la inserción y la fuerza de la manipulación de la recopilación de datos. </li><li> Durante la proyección de imagen funcional de 10 minutos, girar la aguja de 2 minutos en M1 y M2, y deja en su lugar durante los períodos de descanso R1 (2 minutos)), R2 (3 minutos), R3 (1 min). La acupuntura se asegura de no acercarse o dejar el tema directamente antes o después del periodo de estimulación, ya que hasta que el movimiento podría afectar los resultados. (Paradigma, Fig. 1) <img src="/files/ftp_upload/1190/1190fig1.n.jpg" alt="Figura 1" /> Figura 1. Acupuntura paradigma fMRI. </li><li> Tras la finalización de la estimulación de acupuntura, el acupuntor registros de la fuerza con que la aguja se resiste a la manipulación. Al final de cada ciclo, otro investigador de personal pide al sujeto que grado las sensaciones experimentadas en una escala de 1-10. La lista, dolor, dolor, presión, sensación de plenitud, pesadez, entumecimiento, hormigueo, dolor sordo, un dolor agudo, se basa en la experiencia de la comunidad acupunturista y anteriores datos experimentales en lugar de los cuestionarios del dolor en la literatura. La respuesta psicofísica se clasifica como un típico grupo de deqi y otro grupo de deqi mezclado con un dolor agudo sobre la base de estos registros. </li><li> Habla a los sujetos entre imágenes para asegurarse de que se sientan cómodos y se mantienen despiertos. Si alguna parte de su cuerpo comienza a sufrir calambres o se siente incómodo, el personal de investigación en el sitio deben tomar las medidas apropiadas para aliviar el malestar. Los movimientos generados por el sujeto podría causar corrupción de datos. Sujetos ocasionales puede quedarse dormido durante la exploración, lo que afectaría la respuesta hemodinámica. </li></ol> Parte 7: Procesamiento de Datos <ol><li> Múltiples niveles de análisis se puede ejecutar. Un modelo lineal general usa los periodos de descanso como punto de referencia para determinar el cambio de la señal BOLD% MR en cada voxel del cerebro durante la tarea. También determina la significación estadística de estos cambios y elimina los datos BOLD que no llegan a tres voxels contiguos a un determinado valor-p. </li><li> Realizar base de semillas análisis de correlación cruzada para mostrar la conectividad funcional entre una región neurológicos y el resto del cerebro. Esto se realiza mediante la selección de una región de referencia y la comparación de su tiempo-por supuesto con la de cada voxel otros en el cerebro. Otros análisis, como análisis de componentes independientes, análisis de grupos difusos, modelos de ecuaciones estructurales, de causalidad de Granger se puede aplicar. </li><li> Los datos fisiológicos puedenser analizados por un promedio de respuesta dentro de las ventanas relacionadas con la estimulación de agujas de acupuntura. Por ejemplo, los datos de ECG se pueden anotar para la sincronización de los complejos QRS seguido por el análisis de la frecuencia cardiaca y la variabilidad del ritmo cardíaco. </li></ol> Parte 8: Resultados <img src="/files/ftp_upload/1190/1190fig2.n.jpg" alt="Figura 2" /> Figura 2. Respuesta hemodinámica en la acupuntura deqi vs estimulación táctil de la derecha LI4, ST36 y LV3 (52 runs/37 sujetos). Los grupos de estructuras desactivado en el (la corteza prefrontal medial) MPFC (1), MPC (corteza parietal medial) (2), MTL (lóbulo temporal medial) (3), fueron más prominentes en la acupuntura (figura A) que en la estimulación táctil (Fig. B). Desactivación amplia del derecho BA22/21 (4) en la acupuntura no se observó en la estimulación táctil. El BA43 sensoriomotora (5) y la corteza de asociación (6) mostraron una mayor activación de la estimulación táctil que en la acupuntura (no mostrado). Curiosamente, el derecho de la ínsula anterior, (7) se activó en la acupuntura y no en la estimulación táctil. (P &lt;0,0001). <img src="/files/ftp_upload/1190/1190fig3.n.jpg" alt="Figura 3" /> Figura 3. Respuesta hemodinámica en deqi (52 runs/37 sujetos) frente a deqi mezclado con dolor agudo (52 runs/29 sujetos) durante la estimulación de acupuntura a la derecha LI4, ST36 y LV3, la desactivación prominente de la MPFC (1), MPC ( 2) y MTL (3) visto con dolor deqi ausente (Fig. A) fue atenuada en la presencia de dolor (figura B). Con el dolor, la activación de la corteza sensomotora y de asociación (4) se hizo más prominente, y un subconjunto de las regiones límbicas y paralímbicas como el MDIC / SMA (5), postC_BA23 (6), Amy (7), y el vermis cerebeloso ( 8) se activó. El derecho ínsula anterior (9) se activó notablemente en deqi mientras que áreas más pequeñas de la ínsula anterior y posterior (10) se activa de forma bilateral en el dolor. P &lt;0,0001. <img src="/files/ftp_upload/1190/1190fig4.n.jpg" alt="Figura 4" /> Figura 4 Superposición de límbico-neocortical paralímbicas-red (LPNN) en la acupuntura con la red de modo por defecto (DMN). La respuesta BOLD de la LPNN por GLM análisis (Fig. A) y por el modelo libre de análisis de agrupamiento Fuzzy (Fig. B ) durante la estimulación de acupuntura en LI4, ST36 y LV3 muestran patrones casi idénticos a los DMN (Fig. C, adaptado de Shulman et al, 1997). Grupos de regiones con una disminución de la señal coherente aparecen en la corteza prefrontal medial (1), postero-medial corteza (2) y el lóbulo 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>

El empleo de técnicas adecuadas, la experiencia del acupunturista y un tamaño de muestra grande para permitir la separación de los efectos de confusión de la estimulación nociva inadvertida son importantes en la adquisición e interpretación de la acupuntura datos de la fMRI. Nuestros estudios de varios puntos de acupuntura clásicos indican que la acupuntura modula la red límbico, un importante sistema de regulación intrínseca del cerebro humano. La red muestra un solapamiento significativo con el modo por def...

monitoring acupuncture effects on human brain by fmri

La resonancia magnética funcional se utiliza para estudiar los efectos de la acupuntura en la respuesta BOLD y la conectividad funcional del cerebro humano. Los resultados demuestran que la acupuntura moviliza una red límbico-neocortical paralímbicas-y su anti-correlación sensomotora / paralímbicas red en múltiples niveles del cerebro y que la respuesta hemodinámica se ve influenciada por la respuesta psicofísica. Monitorización fisiológica se puede realizar para explorar la respuesta periférica de la función del sistema nervioso autónomo. Este video describe los estudios realizados en LI4 (Hegu), ST36 (Zusanli) y LV3 (Taichong), los puntos de acupuntura clásicos que se utilizan habitualmente para las acciones moduladoras del dolor y la reducción. Algunas cuestiones que requieren atención en las aplicaciones de la resonancia magnética a la investigación de la acupuntura se observan.

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

Monitoreo FMRI y fisiológicos se utiliza para estudiar los efectos de la acupuntura en los sistemas nerviosos central y periférico. La acupuntura moviliza una red límbico-neocortical paralímbicas-, con grandes coincidencias con la red de modo predeterminado, para modular la actividad neurológica, posiblemente relacionado con su efecto autónomo en el sistema nervioso periférico.

Efectos de la acupuntura en vigilancia por fMRI del cerebro humano

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 vistas.  Massachusetts General Hospital and Harvard Medical School. Monitoreo FMRI y fisiológicos se utiliza para estudiar los efectos de la acupuntura en los sistemas nerviosos central y periférico. La acupuntura moviliza una red límbico-neocortical paralímbicas-, con grandes coincidencias con la red de modo predeterminado, para modular la actividad neurológica, posiblemente relacionado con su efecto autónomo en el sistema nervioso periférico.

Video: 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.

Los estudios genéticos de proteínas humanas de reparación del ADN utilizando la levadura como un sistema modelo

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

Investigación

Biología

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.

El análisis de la función de las pequeñas GTPasas de la microinyección de plásmidos en células epiteliales polarizadas

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.

Mapeo de las secuelas de la theta ráfaga de estimulación sobre la corteza auditiva humana con imágenes funcionales

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.

El examen de<em&gt; Drosophila</em&gt; Larvas células traqueales de terminales por microscopía 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 para la Evaluación de los efectos de un rango de longitudes de onda e intensidades de / Terapia de luz del infrarrojo cercano Red sobre el estrés 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.

Los métodos para la comparación de Nutrientes en jalea real Realizado por africanizadas y europeas Honey Bees y los efectos sobre títulos hemolinfa de proteínas

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.

La cuantificación de la adhesividad de la célula 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.

Monitoreo de la homeostasis del calcio del retículo endoplasmático Usando una<em&gt; Gaussia</em&gt; Luciferasa 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.

El aislamiento de regiones genómicas específicas e identificación de moléculas asociadas por 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.

Análisis de glucocorticoides fecales: Monitoreo suprarrenal no invasiva en los équidos

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