Introdução Organismos deve apresentar uma grande variedade de comportamentos para responder a estímulos ameaçadores e mudanças no ambiente. A resposta autonômica é bem estudada em vertebrados como a luta ou fuga resposta em que o sistema nervoso simpático fisiologicamente prepara o ambiente interno (Carpenter, 1976;. Nicholls et al, 2001). É através de alterações fisiológicas que o organismo pode regular a resposta ao estresse e alterar o comportamento de reagir a um ataque iminente ou mudanças ambientais. As evidências mostram que o sistema cardio-vascular está intimamente ligada com o sistema respiratório, assim, permitindo que o ambiente interno para responder e implementar uma resposta. É sabido que o controle autonômico do sistema respiratório e cardiovascular pode regular a disponibilidade de oxigênio e nutrientes para os tecidos-alvo específicos necessários para uma resposta iminente comportamentais. Nos vertebrados, a resposta autonômica e fisiológicos é tão bem evoluído, mas parece provável que os invertebrados complexos, de modo semelhante possuem um sistema de resposta desenvolvidos (Schapker et al, 2002;. Zavarzin, 1941). Apesar de vertebrados e invertebrados são muito diferentes sistematicamente; invertebrados altamente desenvolvidos exigem a rápida resposta cardiovascular e respiratória para responder em uma briga ou modo de vôo. Para muitos invertebrados, uma resposta simpática-como fisiológicos podem ser quantificados. Datado de 1927, muitos dos estudos muito cedo foram realizados em invertebrados, especificamente utilizando artrópodes em geral (Alexandrowicz, 1932; Orlov, 1927; Zavarzin, 1941). Lagostas são conhecidos por apresentar uma ampla gama de comportamentos rápida, assim como a capacidade de avaliar e responder aos estímulos ambientais. Estudos muito cedo notaram uma resposta simpática-como de uma resposta rápida e imediata de postura de defesa (Bethe, 1897; Huxley, 1880; Shuranova et al, 2006;. Wiersma, 1961). O coração lagostas adultas é neurogênica desde a batida eo ritmo são controladas pelo sistema nervoso central (Alexandrowicz, 1932; Yamagishi e Hirose, 1997;. Yamagishi et al, 1997; Wilkens, 1999). Rotineiramente, a medição da taxa de um animal cardíaca (FC) fornece uma medida direta da excitação e prontidão do ambiente interno. Em lagostas, um aumento da FC é notado durante uma postura de defesa quando apresentados com um estímulo percebido ameaçadora (Listerman et al., 2000). Este tipo de resposta do sistema nervoso também foi mostrado em lagostas (Yazawa e Katsuyama, 2001). O sistema respiratório também é neuronalmente controlados com um gerador de padrão ventilatório central (VPG), responsável pela captação de oxigênio através das brânquias pela ação de bombeamento de dois apêndices especializados scaphognathites denominado (Mendelson, 1971). A única scaphognathite reside em cada câmara branquial nas extremidades anterior e tira água através das brânquias por um movimento rítmico (Pasztor, 1968). O VPG, como com o coração, pode ser modulada por diversos fatores. Trabalho anterior no caranguejo, Cancer magister mostra ritmo cardíaco e respiratório podem ser alteradas por controle central através dos neurônios de comando (Wilkens et al., 1974). Além disso, a atividade da VPG é conhecido por mudar com as mudanças na resposta interna e com as interações sociais ou mudanças ambientais em crustáceos (Burmistrov e Shuranova, 1996; cuadras, 1979, 1980; Li et al, 2000;. Listerman et al. , 2000; McMahon e Wilkens, 1983; Schapker et al, 2002;. Shuranova et al, 2002;. Wilkens, 1976). Como visto no crustáceo, atividade ventilatória (RV) pode variar dependendo do estado interno e mudanças no VR pode ser gravado durante inesperados estímulos externos (Shuranova et al, 1993;. Shuranova et al, 2002).. Devido à confiabilidade e viabilidade de RH e medidas VR, as questões futuras a serem feitas são infinitas. Como observado anteriormente, o trabalho tem sido feito para examinar a resposta autonômica durante as interações sociais e distúrbios ambientais. Curiosamente, muitas áreas são deixadas para serem explorados como o exame da resposta autonômica durante alterações hormonais naturais e / ou outros processos fisiológicos que ocorrem no organismo. Possíveis direções futuras também podem examinar simpática-como respostas durante o acasalamento e recepção química olfativa. Mais importante ainda, respostas fisiológicas podem fornecer insights maior em uma resposta do organismo ao meio ambiente quando comparado com as observações comportamentais já que os animais nem sempre respondem comportamentalmente quando eles estão se preparando internamente. Assim, a medida fisiológica fornece um índice biológico para avaliar o estado interno de um organismo que pode explicar as mudanças globais em um organismo. Métodos Antes de começar a fio um crustáceo, preparação de fios é necessária. Corte dois fios de aço inoxidável (diâmetro 0,005 polegadas e com o revestimento 0,008 polegadas; AM Systems, Carlsburg, WA) o tempo suficiente para inserir o lagostim e adequadamente chegar a um detector de impedância (UFI, modelo 2991). Remova o isolamento (~ 0,5 mm) pelo fogo nas extremidades de ambos os fios que serão inseridos na carapaça. Tenha cuidado para não remover o isolamento muito como um fio desencapado na parte externa do animal deve ser recoberto para evitar curto-circuito de impedância gravações. Em seguida, usando uma pinça dobrar as extremidades dos fios queimados em um ângulo de 90 para a colocação na carapaça. Certifique-se que a parte inserida do fio não é muito longa, porque isso poderia causar danos aos órgãos internos. De viabilidade de inserir os fios em um tubo de plástico (que protege os fios), o melhor é vagamente torcer os fios juntos. Se o coração de gravação e as taxas de ventilação, repita os passos acima a uma segunda vez e os dois conjuntos de fios pode ser vagamente trançados juntos antes da colocação no tubo de plástico. Certifique-se de designar os fios para o coração e taxa de ventilação de modo a não confundir os quais os fios que atribuem ao detector de impedância para a gravação. É melhor para rotular os fios cortados ou um par mais curto do que o outro no começo. Viabilizar e segura o fio lagostas, é melhor para embrulhar as garras e as pernas (deixando as costas e um lado exposto), em pano molhado ou toalhas de papel para eliminar o dano à pessoa e / ou lagostim (Figura 1). <img src="/files/ftp_upload/1594/1594fig1.jpg" style="float:left;" alt="Figura 1" /> Figura 1. Envolvimento da chelipeds e as pernas do crustáceo. Envolvendo o crustáceo em uma toalha de papel molhado vai evitar lesões à pessoa e os lagostins. A embalagem vai dificultar a capacidade do crustáceo para mover o chelipeds e pernas. Para iniciar o processo de fiação, é melhor começar na câmara branquial vez que a espessura da carapaça varia de acordo com cada lagostas e força em excesso pode causar danos aos órgãos internos, assim, começando na câmara branquial, a pressão excessiva é menos provável de matar o crustáceo como em comparação com a caixa torácica. O primeiro buraco é feita usando uma ponta fina bisturi e grande o suficiente para um fio para ser colocado debaixo da cutícula na região rostral da câmara branquial (ie, câmara prebranchial para monitorar freqüência ventilatória;. Schapker et al, 2002). Um grande buraco terá perda de hemolinfa excessiva e não veda adequadamente e isso vai aumentar a probabilidade de os fios não permanecendo no buraco designado. Uma vez que o fio está no lugar, queda de uma pequena quantidade de cola (éster de cianoacrilato) e acelerador (HobbyTown EUA, Lexington, KY) para o fio. O uso da cola de secagem rápida reduz o stress manejo dos animais. Tenha cuidado para colocar apenas uma pequena quantidade de acelerador para a cutícula uma vez que este é tóxico para o lagostim e causar a morte. É melhor acabar imediatamente qualquer acelerador adicional fora com papel toalha. Repita essas etapas para o segundo fio na câmara branquial. Estes dois fios são colocados para abranger o scaphognathite (órgão respiratório) para garantir uma medida de impedância forte precisas durante cada movimento scaphognathite (Figura 2). Para obter um registro mais preciso, certifique-se que o segundo fio não é colocado fora da câmara branquial e na caixa torácica. <img src="/files/ftp_upload/1594/1594fig2.jpg" style="float:left;" alt="Figura 2" /> Figura 2. Colocação de fios de aço inoxidável na câmara branquial. Os pontos brancos na carapaça representam pontos para os fios de aço inoxidável para ser colocado sob a cutícula. Os fios atravessam o scaphognathite e movimento através da medida de impedância dinâmica. Os passos para a taxa de fiação coração na caixa torácica é o mesmo que acima, exceto os fios são colocados sob a carapaça dorsal diretamente sobre o coração e extensão do coração em um arranjo rostral-caudal (Figura 3). Este posicionamento garante uma medida de impedância forte precisas durante cada contração do coração (Listerman et al., 2001). <img src="/files/ftp_upload/1594/1594fig3.jpg" style="float:left;" alt="Figura 3" /> Figura 3. Colocação de fios de aço inoxidável na caixa torácica. Os pontos brancos na carapaça representam pontos para os fios de aço inoxidável para ser colocado sob a cutícula. Os fios atravessam o coração em um arranjo rostral-caudal. Isto irá permitir a medidas de contração do coração através de impedância dinâmica. Uma vez que a colocação de todos os quatro fios está completo, misturar um adesivo instantâneo (Eastman, 5 min e secagempoxy) e revestimento dos furos para cada local, assim como os fios. Uma vez que os fios e furos sejam adequadamente cobertos, use o epóxi restantes para segurar o tubo de plástico à volta do lagostim. Isso irá garantir que os fios não vai ser puxado para fora dos buracos em manejos futuros. Desde o epóxi leva alguns minutos para secar, deter ou coloque o crustáceo em um recipiente sem água até que o epoxy não é mais pegajosa ao toque. Uma vez que o epoxi é seco, lavar as lagostas em água para remover excesso de produtos químicos e colocá-los de volta em um ambiente aquático. Para garantir a saúde do crustáceo, o melhor é trocar a água em cerca de uma hora para remover quaisquer substâncias químicas não previamente removido. Como se observa dos investigadores anteriores, descobrimos que manipulação prolongada altera a medidas fisiológicas de estresse, devido por um período de tempo de 24 h para 3 dias (Wilkens et al, 1985.), Portanto, os animais devem ser deixados para 2 a 3 dias antes de qualquer experimentação . ECGs para gravar, conectar os fios do crustáceo a detectores de impedância. Os dois fios que medem o cardíaca (FC medida) deve ser ligado a um detector de impedância único e mesmo para os dois fios que medem o scaphognathite (medida VR). O detector de impedância medidas resistência dinâmica entre os dois fios de aço inoxidável. Esses sinais são captados por ligar o detector on-line para um PowerLab através de uma interface PowerLab/4SP (Instrumentos AD). Todos os eventos são medidos e calibrado com a versão do software 5.5.6 Gráfico PowerLab (AD Instruments, Austrália). Para começar, defina uma taxa de aquisição de 4 kHz. As deflexões grandes representará batimento cardíaco ea taxa de ventilação. Para facilitar a contagem de cada HR / VR, uma função de suavização pode aplicar ao conjunto de dados. Se 100 pontos foram suavizados (isto é, em média) o traço resultante vai retratar apenas os batimentos cardíacos. A freqüência cardíaca pode ser determinado por contagem direta de cada batida mais de 10 s intervalos e depois convertido em batimentos por min (BPM).

<ol>
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Zool. 46, 585-596 (1968).</li><li>Schapker, H., Breithaupt, T., Shuranova, Z., Burmistrov, Y., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+rate+and+ventilatory+correlative+measures+in+crayfish+during+environmental+disturbances+and+social+interactions.">Heart rate and ventilatory correlative measures in crayfish during environmental disturbances and social interactions.</a> Comp. Biochem. Physiol., A. 131, 397-407 (2002).</li><li>Shuranova, Z. P., Vekhov, A. V., Burmistrov, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behavioral+reactions+of+fresh-water+crayfish+to+sensory+exposures:+the+autonomic+components.">The behavioral reactions of fresh-water crayfish to sensory exposures: the autonomic components.</a> Zh. Vyssh Nerv. 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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

A resposta de vôo medo, ou lutar serve de base fisiológica fundamental para examinar a consciência de um organismo de seu ambiente em um ataque predador iminente. Embora não se sabe se os invertebrados possuem um sistema nervoso autônomo idêntico ao dos vertebrados, a evidência mostra invertebrados têm uma resposta simpática-como para regular o ambiente interno e preparar o organismo para agir comportamental a um estímulo dado. Além disso, essa resposta fisiológica pode ser viável medidos e ele age como um índice biológico para o estado interno do animal. Medições da resposta fisiológica podem estar diretamente relacionados a estressores internos e externos através de mudanças na coordenação do sistema nervoso central controlada dos sistemas cardio-vascular e respiratória. Mais especificamente, a monitorização cardíaca e ventilação fornecer medidas quantificáveis ​​da resposta ao estresse nem sempre comportamentais observadas. Lagostas são organismos bom modelo para o coração e medições freqüência ventilatória devido à possibilidade de gravação, bem como a rica história conhecida da morfologia do crustáceo, que remonta a Huxley em 1888, e os comportamentos bem estudado típico.

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 apresentam uma resposta simpática-como autônomo semelhante ao descrito para os vertebrados. A coordenação dos sistemas cardio-vascular e ventilatórias permite a medição de um índice biológico no qual se pode avaliar um estado organismo interna.

Medidas de freqüência cardíaca e ventilatória em se movimentar livremente Crayfish

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 visualizações.  University of Kentucky. Invertebrados apresentam uma resposta simpática-como autônomo semelhante ao descrito para os vertebrados. A coordenação dos sistemas cardio-vascular e ventilatórias permite a medição de um índice biológico no qual se pode avaliar um estado organismo interna.

Vídeo: 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.

Implantação de engenharia de tecidos no coração de ratos

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

Biologia

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.

Gravações da ativação do circuito neural no Livremente Behaving Animais

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

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

Visualizar o coração batendo no Drosophila</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

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 e demonstração Fisiologia da MNJ da Muscle Opener Crayfish

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.

Preparação de embriões de Microscopia Eletrônica do Drosophila Tubo cardíaco embrionário

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 &amp;#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.

Quantificação de γH2AX Focos em resposta à radiação 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 ...

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 &amp;#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.

Avaliação da Distribuição Espacial da γH2AX seguintes Radiação Ionizante

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 ...

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.

Avaliação do cálcio Faíscas intactas 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.

Medição do mRNA Decay Preços em<em&gt; Saccharomyces cerevisiae</em&gt; Usando<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 ...

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by &#946;-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization of the ryanodine receptor, a homo-tetrameric ion channel mediating Ca2+ release from intracellular stores, is critical for skeletal and cardiac muscle contraction. Here, we present complementary in vivo and in vitro methods to detect protein self-association and determine homo-oligomer stoichiometry.

Abordagens genéticas e bioquímicas para<em&gt; In Vivo</em&gt; e<em&gt; In Vitro</em&gt; Avaliação de oligomerização da proteína: A Case Study rianodina Receptor

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ...