Introducción Los organismos deben presentar una amplia gama de comportamientos que responden a los estímulos de amenaza y los cambios en el medio ambiente. La respuesta autonómica está bien estudiado en los vertebrados como la respuesta de lucha o de vuelo en el que el sistema nervioso simpático prepara fisiológicamente el ambiente interno (Carpenter, 1976;. Nicholls et al, 2001). Es a través de los cambios fisiológicos que un organismo puede regular la respuesta al estrés y modificar la conducta de reaccionar ante un ataque inminente o cambios ambientales. La evidencia muestra que el sistema cardio-vascular está estrechamente conectada con el sistema respiratorio permitiendo así que el ambiente interno de responder y ejecutar una respuesta. Es bien sabido que el control autónomo de los sistemas respiratorio y cardiovascular pueden regular la disponibilidad de oxígeno y nutrientes a los tejidos específicos necesarios para una respuesta de comportamiento inminente. En los vertebrados, la respuesta autonómica y fisiológico está tan bien desarrollado, parece probable que los invertebrados complejos de manera similar que poseen un sistema de respuesta desarrollados (Schapker et al, 2002;. Zavarzin, 1941). A pesar de que los vertebrados y los invertebrados son muy diferentes de forma sistemática; invertebrados muy desarrollados requieren la rápida respuesta cardiovascular y respiratoria para responder de una forma de lucha o huida. Para muchos invertebrados, una respuesta fisiológica simpático-como se puede cuantificar. Que data de 1927, muchos de los estudios muy temprano se han realizado en los invertebrados, especialmente artrópodos, en general, utilizando (Alexandrowicz, 1932; Orlov, 1927; Zavarzin, 1941). Cangrejos de río se sabe que presentan una amplia gama de comportamientos rápida, así como la capacidad para evaluar y responder a los estímulos ambientales. Estudios muy temprano han observado una respuesta simpática, como de una respuesta inmediata y rápida de la postura de defensa (Bethe, 1897; Huxley, 1880; Shuranova et al, 2006;. Wiersma, 1961). El corazón del cangrejo de río adulto neurogénica desde el compás y el ritmo son controlados por el sistema nervioso central (Alexandrowicz, 1932; Yamagishi y Hirose, 1997;. Yamagishi et al, 1997; Wilkens, 1999). Habitualmente, la medición de la frecuencia cardiaca de un animal (HR) proporciona una medida directa de la excitabilidad y la preparación del ambiente interno. En cangrejos de río, un incremento de la FC se observa en una postura de defensa cuando se le presenta un estímulo percibido amenaza (Listerman et al., 2000). Este tipo de respuesta del sistema nervioso también se mostró en las langostas (Yazawa y Katsuyama, 2001). El sistema de ventilación es también neuronal controlado con un generador de patrón ventilatorio central (VPG) responsable de la captación de oxígeno través de las branquias por la acción de bombeo de dos apéndices especializados denominados scaphognathites (Mendelson, 1971). A scaphognathite solo reside en cada cámara branquial en los extremos anterior y lleva el agua a través de las branquias de un movimiento rítmico (Pasztor, 1968). El VPG, al igual que con el corazón, puede ser modulado por muchos factores. El trabajo previo en el cangrejo, Cancer magister muestra el ritmo cardíaco y respiratorio puede ser alterado por el control central a través de las neuronas de comando (Wilkens et al., 1974). Además, la actividad de la VPG se conoce a cambiar con los cambios en la respuesta interna y con las interacciones sociales o los cambios ambientales en los crustáceos (Burmistrov y Shuranova, 1996; Cuadras, 1979, 1980, Li et al, 2000;. Listerman et al. , 2000; McMahon y Wilkens, 1983; Schapker et al, 2002;. Shuranova et al, 2002;. Wilkens, 1976). Como se observa en los cangrejos de río, la actividad ventilatoria (VR) puede variar dependiendo de la situación interna y los cambios en la realidad virtual se pueden registrar durante inesperados estímulos externos (Shuranova et al, 1993;. Shuranova et al, 2002).. Debido a la seriedad y viabilidad de los recursos humanos y las medidas de realidad virtual, las preguntas que se le pregunte el futuro son infinitas. Como se señaló anteriormente, el trabajo se ha hecho para examinar la respuesta autonómica durante las interacciones sociales y las alteraciones del medio ambiente. Curiosamente, muchas áreas quedan por explorar, como el examen de la respuesta autonómica durante los cambios hormonales naturales y / o en otros procesos fisiológicos que ocurren en el organismo. Las posibles direcciones futuras también puede examinar simpático-como las respuestas durante el apareamiento y la recepción química olfato. Más importante aún, las respuestas fisiológicas pueden proporcionar un mayor conocimiento de una respuesta del organismo al medio ambiente en comparación con las observaciones de la conducta ya que los animales no siempre se comportaron cuando se están preparando internamente. Por lo tanto, la medición fisiológica proporciona un índice biológico para evaluar el estado interno de un organismo que puede explicar los cambios globales en el organismo. Métodos Antes de comenzar el hilo de un cangrejo de río, la preparación de los cables es necesario. Cortar dos cables aislados de acero inoxidable (diámetro de 0,005 pulgadas y con el revestimiento de 0,008 pulgadas, sistemas de AM, Carlsburg, WA) el tiempo suficiente para insertar en los cangrejos de río y adecuadamente llegar a un detector de impedancia (UFI, modelo 2991). Quite el aislamiento (~ 0,5 mm) por el fuego en los extremos de los dos cables que se insertan en el caparazón. Tenga cuidado de no remover demasiado aislamiento como un cable pelado en el exterior del animal deben ser recubiertas para evitar cortocircuitos en las grabaciones de impedancia. Entonces, el uso de fórceps doblar los extremos de los cables quemados en un ángulo de 90 para la colocación en el caparazón. Asegúrese de que inserta la porción del cable no es demasiado largo, porque esto podría hacer daño a los órganos internos. Para la viabilidad de la inserción de los cables en un tubo de plástico (que protege los cables), lo mejor es girar libremente los cables juntos. Si el corazón de grabación y tasas de ventilación, repita los pasos anteriores por segunda vez y los dos juegos de cables puede ser vagamente trenzado juntos antes de la colocación en el tubo de plástico. Asegúrese de designar a los cables para el corazón y la tasa de ventilación a fin de no confundir los cables que unen a detector de impedancia, que para la grabación. Lo mejor es la etiqueta de los cables o cortar un par más corto que el otro en el principio. Para factible y segura de alambre del cangrejo de río, lo mejor es envolver las garras y las piernas (dejando la parte de atrás y se expone uno de los lados) en un paño húmedo o toallas de papel para eliminar el perjuicio a la persona y / o cangrejos de río (Figura 1). <img src="/files/ftp_upload/1594/1594fig1.jpg" style="float:left;" alt="Figura 1" /> Figura 1. Envoltura del quelípedos y las piernas del cangrejo de río. Envolver los cangrejos de río en una toalla de papel húmeda evitará lesiones a la persona y el cangrejo. El envoltorio se obstaculizan la capacidad de los cangrejos de río para mover la quelípedos y las piernas. Para comenzar el proceso de cableado, lo mejor es empezar en la cámara de enmalle ya que el grosor del caparazón varía con cada uno de los cangrejos y exceso de fuerza puede causar daños en órganos internos, por lo que al comenzar en la cámara de enmalle, la presión excesiva es menos probable que matar a los cangrejos de río como en comparación con la cámara torácica. El primer agujero se realiza mediante un bisturí de punta fina y grande suficiente para un alambre que se inserta debajo de la cutícula de la zona rostral de la cámara de enmalle (es decir, la cámara prebranchial para controlar la frecuencia respiratoria;. Schapker et al, 2002). Un gran agujero tendrá la pérdida de hemolinfa excesiva y no sellar correctamente y que esto aumentará la probabilidad de que los cables no quedan en el agujero designado. Una vez que el cable está en su lugar, colocar una pequeña cantidad de pegamento (éster de cianoacrilato) y el acelerador (HobbyTown EE.UU., Lexington, KY) en el cable. El uso del pegamento de secado rápido, reduce el estrés del manejo de los animales. Tenga cuidado de colocar sólo una pequeña cantidad de acelerador en la cutícula ya que es tóxico para los cangrejos de río y causar la muerte. Lo mejor es borrar inmediatamente cualquier acelerador sobrante con una toalla de papel. Repita estos pasos para el segundo cable en la cámara de las agallas. Estos dos cables se colocan para abarcar el scaphognathite (órgano respiratorio) para asegurar una fuerte medida de impedancia correcta en cada movimiento scaphognathite (Figura 2). Para obtener un registro exacto, asegúrese de que el segundo hilo no se coloca fuera de la cámara branquial y en la cámara torácica. <img src="/files/ftp_upload/1594/1594fig2.jpg" style="float:left;" alt="Figura 2" /> Figura 2. La colocación de alambres de acero inoxidable en la cámara de las agallas. Los puntos blancos en el caparazón representan puntos para los alambres de acero inoxidable que se coloca debajo de la cutícula. Los cables de abarcar el scaphognathite y el movimiento a través de medidas de impedancia dinámica. Los pasos para la tasa de cableado de corazón en la cámara de tórax es el mismo que el anterior, excepto los cables se colocan bajo el caparazón dorsal directamente sobre el corazón y la duración del corazón en un acuerdo de rostral-caudal (Figura 3). Esta colocación se asegurará una fuerte medida de impedancia exacta durante cada contracción del corazón (Listerman et al., 2001). <img src="/files/ftp_upload/1594/1594fig3.jpg" style="float:left;" alt="Figura 3" /> Figura 3. La colocación de alambres de acero inoxidable en la cámara torácica. Los puntos blancos en el caparazón representan puntos para los alambres de acero inoxidable que se coloca debajo de la cutícula. Los cables abarcan todo el corazón en un acuerdo de rostral-caudal. Esto permitirá a las medidas de la contracción del corazón a través de impedancia dinámica. Una vez que la colocación de los cuatro cables es completa, mezcla de un adhesivo instantáneo (Eastman, de 5 minutos de secado epoxy) y el abrigo de los agujeros de cada lugar, así como los cables. Una vez que los cables y los agujeros están adecuadamente cubiertas, utilice la resina restante para mantener el tubo de plástico para la parte de atrás de los cangrejos de río. Esto asegurará que los cables no se retiró de los agujeros en los manejos en el futuro. Dado que la resina tarda unos minutos para secar, bodega o lugar de los cangrejos en un recipiente sin agua hasta que el epoxi ya no es pegajoso al tacto. Una vez que la resina se seca, lavar los cangrejos de río con agua para eliminar exceso de productos químicos y colocarlos de nuevo en un ambiente acuático. Para asegurar la salud de los cangrejos de río, lo mejor es cambiar el agua de aproximadamente una hora para eliminar cualquier sustancia química no previamente eliminado. Como se ha señalado a los investigadores anteriores, encontramos que la manipulación prolongada altera las mediciones de estrés fisiológico debido a una duración de 24 horas a 3 días (Wilkens et al, 1985.), Por lo tanto, los animales se debe dejar de 2 a 3 días antes de la experimentación . Para grabar ECGs, conecte los cables de los cangrejos de río a los detectores de impedancia. Los dos cables que abarca el corazón (HR medida) debe ser conectado a un detector de impedancia de un solo y mismo para los dos cables que abarca el scaphognathite (medida VR). El detector de impedancia mide la resistencia dinámica entre los dos alambres de acero inoxidable. Estas señales son captadas por el detector de conectar en línea a una PowerLab través de una interfaz PowerLab/4SP (Instrumentos AD). Todos los eventos son medidos y calibrados con la versión de software PowerLab Gráfico 5.5.6 (AD Instruments, Australia). Para empezar, establezca una velocidad de adquisición de 4 kHz. Las desviaciones grandes se representan latidos del corazón y la tasa de ventilación. Para facilitar el conteo de cada uno de HR / VR, se puede aplicar una función de suavizado de los datos. Si 100 puntos se suavizaron (es decir, en promedio) la huella resultante se muestran sólo los latidos del corazón. La frecuencia cardíaca puede ser determinada por conteo directo de cada latido de más de 10 s intervalos y luego se convierte en latidos por minuto (BPM).

<ol>
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<table border="1">
<tbody>
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
</tr>	
<tr>
<td>Scalpel (Stainless steel surgical blades No. 11 for No. 3 handle)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Stainless steel wire (diameter 0.005 inches with the coating 0.008 inches; A-M Systems, Carlsburg, WA)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Impedance detector (UFI, model 2991)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Plastic tubing (Tygon Tubing ID .0812, OD .1492, Cole-Parmer Instrument Company)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Fast-drying glue (cyanoacrylate ester, HobbyTown USA, Lexington, KY)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Accelerator (HobbyTown USA, Lexington, KY)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Adhesive (Eastman, 5-min drying epoxy)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td><a href="http://www.adinstruments.com/products/hardware/corporate/product/ML866/" target="_blank">PowerLab via a PowerLab/4SP interface (AD Instruments)</a>.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td><a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank">PowerLab Chart software version 5.5.6 (AD Instruments, Australia)</a></td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td><a href="http://www.adinstruments.com/products/software/research/LabChart-Software/" target="_blank">Chart software version 3.5.6 (AD Instruments, Australia)</a></td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
</tbody>
		</table>

measures of heart and ventilatory rates in freely moving crayfish

La respuesta al miedo, la huida o la lucha es la base fisiológica fundamental para el examen de conciencia de un organismo de su entorno en un ataque de depredadores inminente. Aunque no se sabe si los invertebrados poseen un sistema nervioso autónomo, idéntica a la de los vertebrados, la evidencia muestra los invertebrados tienen una respuesta simpática, como para regular el medio interno del organismo y listo para actuar conductualmente a un estímulo determinado. Además, esta respuesta fisiológica puede ser factible medir y actúa como un índice biológico para el estado interno del animal. Las mediciones de la respuesta fisiológica puede estar directamente relacionado con factores estresantes internos y externos a través de cambios en la coordinación del sistema nervioso central controlado de los sistemas cardiovascular y respiratorio. Más específicamente, el monitoreo cardíaco y las tasas de ventilación proporcionan mediciones cuantificables de la respuesta al estrés no siempre su comportamiento observado. Cangrejos de río son organismos buen modelo para el corazón y las mediciones de tasa de ventilación debido a la posibilidad de grabación, así como la rica historia conocida de la morfología de los cangrejos de río, que data de Huxley en 1888, y las conductas típicas bien estudiado.

Watch this Scientific Journal Video about Measures of Heart and Ventilatory Rates in Freely Moving Crayfish at JoVE.com

Measures of Heart and Ventilatory Rates in Freely Moving Crayfish

Invertebrados muestran un autónomo simpático-como respuesta similar a la descrita para los vertebrados. La coordinación de los sistemas cardiovascular y respiratorio permite la medición de un índice biológico en el que para evaluar un estado de organismo interno.

Medidas de las frecuencias cardíaca y respiratoria en movimiento libre del cangrejo de río

The fear, flight or fight response serves as the fundamental physiological basis for examining an organism's awareness of its environment under an ...

measures-of-heart-and-ventilatory-rates-in-freely-moving-crayfish

Research

JoVE Journal

Biology

15.2K vistas.  University of Kentucky. Invertebrados muestran un autónomo simpático-como respuesta similar a la descrita para los vertebrados. La coordinación de los sistemas cardiovascular y respiratorio permite la medición de un índice biológico en el que para evaluar un estado de organismo interno.

Video: Measures of Heart and Ventilatory Rates in Freely Moving Crayfish

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Implantación de la ingeniería tisular en el corazón de rata

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Investigación

Biología

Recordings of Neural Circuit Activation in Freely Behaving Animals

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. Traditional non-invasive methods require at least partially restrained research subjects, and they only allow identification of large numbers of simultaneously activated neurons. On the other hand, small ensembles of neurons or individual neurons can only be measured using single-cell recordings obtained from largely reduced preparations. Since the expression of natural behavior is limited in restrained and dissected animals, the underlying neural mechanisms that control such behavior are difficult to identify. 
Here, I present a non-invasive physiological technique that allows measuring neural circuit activation in freely behaving animals. Using a pair of wire electrodes inside a water-filled chamber, the bath electrodes record neural and muscular field potentials generated by juvenile crayfish during natural or experimentally evoked escape responses. The primary escape responses of crayfish are mediated by three different types of tail-flips which move the animals away from the point of stimulation. Each type of tail-flip is controlled by its own neural circuit; the two fastest and most powerful escape responses require activation of different sets of large &#8220;command&#8221; neurons. In combination with behavioral observations, the bath electrode recordings allow unambiguous identification of these neurons and the associated neural circuits. Thus activity of neural circuitry underlying naturally occurring behavior can be measured in unrestrained animals and in different behavioral contexts.

Non-invasive measurements of neural activity patterns in freely behaving animals are obtained by combining neurophysiological recordings with high speed videography.

Grabaciones de la activación de circuitos neuronales en animales comportarse libremente

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. ...

Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is still much to learn from this model preparation. In understanding the history one can appreciate why even today this NMJ still offers a rich playground to address questions regarding pre- and post-synaptic function and plasticity. The viability and ease of access to the terminal for intracellular as well as extracellular electrophysiology and imaging are significant advantages. The mechanisms behind the modulation of vesicular kinetics and fusion within the high- and low-output terminals are begging for investigation. The preparation also offers a testable model system for computational assessments and manipulations to examine key variables in theoretical models of synaptic function, for example calcium dynamics during short-term facilitation. The synaptic complexity of active zone and statistical nature of quantal release is also an open area for future investigation both experimentally and computationally.

The opener muscle of the crayfish leg is presented for its historical importance and experimental versatility in muscle phenotype, synaptic physiology and plasticity.

Ver histórico y demostración de Fisiología en la UNM de los músculos inaugural cangrejo de río

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Preparación de embriones para Microscopía Electrónica de la Drosophila Tubo cardíaco embrionario

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

La cuantificación de γH2AX focos en respuesta a la radiación ionizante

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

La cuantificación de γH2AX focos en muestras de tejido

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

Evaluación de la distribución espacial de la radiación ionizante γH2AX siguientes

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Ensayo clonigénicas: las células adherentes

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Evaluación de calcio en Sparks intactas las fibras musculares esqueléticas

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

La medición de mRNA decadencia Tarifas en<em&gt; Saccharomyces cerevisiae</em&gt; Uso<em&gt; Rpb1-1</em&gt; Cepas

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...