简介生物必须具有广泛的行为，威胁性的刺​​激和环境的变化作出回应。自主的反应是很好的研究脊椎动物在交感神经系统的生理准备的内部环境（木匠，1976年; Nicholls 等人，2001年）的战斗或逃跑反应。这是一个有机体，可以调节应激反应和改变行为，以应对即将发生的袭击或环境变化，通过生理上的变化。有证据表明，心血管系统是紧密连接的呼吸道系统，从而使内部环境的响应和落实响应。这是众所周知的呼吸系统和心血管系统的植物神经控制，可调节氧气供应和即将发生的行为反应所需要的特定的目标组织的营养物质。 在脊椎动物中，自主和生理反应以及演变，它似乎复杂的无脊椎动物，同样也会拥有一个发达的响应系统（Schapker等 ，2002; Zavarzin，1941年）。即使脊椎动物和无脊椎动物有很大的不同，系统高度发达的无脊椎动物需要心血管和呼吸系统的反应迅速，在战斗或飞行的方式回应。对于许多无脊椎动物，一种同情般的生理反应是可以量化的。的历史可以追溯到到1927年，许多非常早期的研究已经进行了无脊椎动物，特别是在一般使用节肢动物（Alexandrowicz，1932年;奥尔洛夫，1927年; Zavarzin，1941年）。 小龙虾被称为表现出广泛的迅速行为，以及评估和应对环境刺激的能力。极早期的研究指出了防御的姿态立 ​​即和快速反应（贝特，1897年，赫胥黎，1880年; Shuranova等，2006; Wiersma，1961年）的同情般的响应。成人小龙虾的心源性中枢神经系统（Alexandrowicz，1932年，山岸和广濑，1997年，山岸等，1997;威尔肯斯，1999年）以来的节拍和节奏的控制。常规动物的心率（HR）的测量提供了一种兴奋和内部环境的准备的直接措施。小龙虾，在人力资源的增加是与预见的威胁刺激（Listerman等 ，2000）提出时指出，在防御的姿态。这种类型的神经系统的反应也显示了刺龙虾（矢泽胜山，2001年）。 通气系统也neu​​ronally一个通气中枢模式发生器（VPG）由两个专门的附属物称为scaphognathites抽水行动（门德尔松，1971年），负责整个鳃摄氧量控制。一个单一scaphognathite驻留在每个鳃腔前结束，并提请整个鳃有节奏的运动（Pasztor，1968）的水。 VPG，与心脏一样，可以由许多因素调制。在以往的工作，螃蟹癌症魔导师显示心脏和换气的节奏，可以通过命令神经元（ 威尔肯斯等人，1974年）的中央控制的改变。此外，VPG的活动被称为改变内部的反应，并与社会互动或甲壳类动物中的环境变化（Burmistrov Shuranova，1996年的变化; Cuadras，1979年，1980年，李等 ， 2000; Listerman等。 ，2000;麦克马洪和威尔肯斯，1983年; Schapker 等 ，2002;。Shuranova 等 ，2002;威尔肯斯，1976年）。在小龙虾，通气活动（VR）可以不同的内部状态，并在VR的变化而定，可以在意想不到的外部刺激（Shuranova等，1993; Shuranova等，2002 。）记录。 由于人力资源和VR措施的可靠性和可行性，未来要问的问题是无穷无尽的。如前所述，工作已经做了研究，在社会交往和环境干扰的自主反应。有趣的是，许多领域都留下探索，如审查期间天然激素的变化和/或其他有机体中发生的生理过程自主反应。在交配和嗅觉的化学接待过程中，今后可能的方向，也可能检查交感神经样的反应。最重要的是，生理反应可能成一个有机体的反应相比，行为观察，因为动物不总是响应行为时，他们正准备自己内部环境提供更大的见解。因此，生理测量提供了一个评估一个有机体的内部状态，这也许可以解释在一个有机体的整体变化的生物指标。 方法才开始连接一个小龙虾，需要准备的电线。切两根绝缘的不锈钢丝（直径为0.005英寸和0.008英寸的涂层;上午系统，Carlsburg，WA），足够长的时间插入到小龙虾，并充​​分达到阻抗探测器（UFI模型2991）。删除将插入到背甲两线两端的防火隔热（〜0.5毫米）。小心不作为裸线删除过多的动物外绝缘必须重涂，以防止短路阻抗录音。然后，使用产钳在一个90度的电线的弯曲放置到甲壳烧毁结束。确定的电线插入部分是不能太长，因为这可能内脏损害。对于插入塑料管（保护导线）线的可行性，它是最好的，以松散的扭曲电线。如果记录心脏和通风率，重复上述步骤，第二次和两套线可以是松散的编织前的位置连成塑料管材。请务必指定为心脏和通风率的电线，因此不会混淆哪一根导线的阻抗探测器附加录制。这是最好的标签上的电线或削减一对，比其他较短的开始。 可行和安全的电线的小龙虾，最好是包裹在潮湿的布或纸巾的爪子和腿（离开的背部和一个侧面暴露），以消除伤害的个人和/或小龙虾（图1）。 <img src="/files/ftp_upload/1594/1594fig1.jpg" style="float:left;" alt="图1" />图1。包装的小龙虾的螯和腿。在湿纸巾包装的小龙虾，避免造成人身伤害的人，小龙虾。包装将阻碍移动能力的小龙虾的螯和腿。 要开始的布线过程，最好是开始在鳃腔，因为每个小龙虾和多余的力量与甲壳厚度不同可引起内脏损害，从而开始在鳃腔，压力过大是不太可能杀的小龙虾而胸室。第一洞是由使 ​​用手术刀和公正的角质层下一个要插入线在鳃腔（即prebranchial室监测通气率; Schapker等，2002）喙面积足够大的细点。一个大洞，将有过多的血淋巴中的损失和不正确密封，这将增加不留在指定的孔线的概率。电线一旦到位，拖放到丝胶（氰基丙烯酸酯）和加速器（HobbyTown美国，列克星敦，肯塔基州）的少量。快速干燥胶的使用，减少动物的压力处理。小心将其放置在角质层，只有少量的加速器，因为这是有毒的小龙虾，并会导致死亡。这是最好立即用纸巾擦拭任何多余的加速器。重复这些步骤，在鳃腔的第二线。这两根线放在跨度scaphognathite（呼吸器官），以确保在每个scaphognathite运动（图2）强大的精确的阻抗测量。为了得到准确的记录，可以肯定的，第二线外鳃腔，并放置到胸腔。 <img src="/files/ftp_upload/1594/1594fig2.jpg" style="float:left;" alt="图2" />图2。放置在鳃腔的不锈钢丝。背甲上的白点，表示为不锈钢角质层下放置的导线点。导线跨越scaphognathite通过动态阻抗和衡量运动。 布线心率胸腔的步骤与上述相同的电线都背背甲下直接放置在心脏和跨度之心（图3）在延髓尾鳍安排的除外。这个位置将确保在每次心脏收缩（Listerman 等 ，2001）强大的精确的阻抗测量。 <img src="/files/ftp_upload/1594/1594fig3.jpg" style="float:left;" alt="图3" />图3。不锈钢丝放置在胸腔。背甲上的白点，表示为不锈钢角质层下放置的导线点。该线跨越在延髓尾鳍安排的核心。这将允许通过动态阻抗心脏收缩的措施。 一旦完成所有四线的位置，混合瞬间粘合剂（伊士曼，5分钟干燥Ëpoxy）和大衣每个位置以及电线孔。一旦有足够的覆盖，电线和钻孔使用剩余的环氧举行塑料管材后面的小龙虾。这将确保不会拉电线在未来handlings的孔。由于环氧树脂需要几分钟，晾干，持有或在一个没有水的容器的地方小龙虾，直到环氧树脂不再粘到触摸。一旦环氧树脂干，小龙虾在水中清洗，以除去多余的化学品，并重新放入水环境。为了确保小龙虾的健康，最好是改变在大约一个小时的水除去以前没有删除任何化学品。从以前的调查中指出，我们发现，长期处理因压力改变了生理测量，持续时间为24小时至3天（威尔肯斯等 ，1985）。;因此，任何实验前动物应留2至3天。 为了记录心电图，连接电线，小龙虾阻抗探测器。应跨越心（衡量人力资源）的两根电线连接到一个单一的阻抗探测器，同样的两根导线跨越scaphognathite（衡量的VR）。阻抗探测器措施，两者之间的不锈钢丝的动态电阻。这些信号记录的检测器连接上线，通过PowerLab/4SP接口（公元仪器）一个PowerLab系统。衡量所有的事件都与PowerLab系统图软件版本5.5.6（公元仪器，澳大利亚）校准。首先，收购率在4千赫。大挠度将代表心跳和通风率。为了便于计数每一个人力资源/ VR，平滑功能可以应用到数据集。如果100点平滑（即平均值）所造成的轨迹，将描绘只有心跳。心跳率可以由每超过10秒的间隔击败直接计数，然后转换为每分钟的节拍（BPM）的。

<ol>
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L., Mantel, L., Bliss, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventilation,+perfusion+and+oxygen+uptake.">Ventilation, perfusion and oxygen uptake.</a> Biology of Crustacea. 6, 289-372 (1983).</li><li>Mendelson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillator+neurons+in+crustacean+ganglia.">Oscillator neurons in crustacean ganglia.</a> Science. 171, 1170-1173 (1971).</li><li>Nicholls, J. G., Martin, A. R., Wallace, B. G., Fuchs, P. 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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. Deiat Im I P Pavlova. 43, 1159-1169 (1993).</li><li>Shuranova, Z. P., Burmistrov, Y. M., Wiese, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventilatory+activity+in+free+moving+crayfish+is+indicative+of+its+functional+state+and+perceiving+external+stimuli.">Ventilatory activity in free moving crayfish is indicative of its functional state and perceiving external stimuli.</a> The Crustacean Nervous System. , 526-535 (2002).</li><li>Shuranova, Z. P., Burmistrov, Y. M., Strawn, J. R., 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=Evidence+for+an+autonomic+nervous+system+in+decapod+crustaceans.">Evidence for an autonomic nervous system in decapod crustaceans.</a> Inter. J. Zool. Res. 2, 242-283 (2006).</li><li>Wiersma, C. A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflexes+and+the+central+nervous+system.">Reflexes and the central nervous system.</a> The physiology of Crustacea, vol II, Sense organs, integration, and behavior. , 241-279 (1961).</li><li>Wilkens, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+control+of+respiration+in+decapod+Crustacea.+Fed.">Neuronal control of respiration in decapod Crustacea. Fed.</a> Fed. Proc. 35, 2000-2006 (1976).</li><li>Wilkens, J. L., Wilkens, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+control+of+cardiac+and+scaphognathite+pacemakers+in+the+crab+Cancer+magister.">Central control of cardiac and scaphognathite pacemakers in the crab Cancer magister.</a> J. Comp. Physiol. 90, 89-104 (1974).</li><li>Wilkens, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+cardiac+rhythmicity+and+of+blood+distribution+in+crustaceans.">The control of cardiac rhythmicity and of blood distribution in crustaceans.</a> Comp. Biochem. Physiol. A. 124, 531-538 (1999).</li><li>Yamagishi, H., Ando, H., Makioka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myogenic+heartbeat+in+the+primitive+crustacean+Triops+longicaudatus.">Myogenic heartbeat in the primitive crustacean Triops longicaudatus.</a> Biol. Bull. 193, 350-358 (1997).</li><li>Yazawa, T., Katsuyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+and+repetitive+cardiac+slowdown+in+the+freely+moving+spiny+lobster,+Panulirus+japonicas.">Spontaneous and repetitive cardiac slowdown in the freely moving spiny lobster, Panulirus japonicas.</a> J. Comp. Physiol. 187, 817-824 (2001).</li><li>Zavarzin, A. A., Zavarzin, A. A., Izbrannye, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ocherki po evol utsionnoj gistologii nervnoj sistemy (Essays on the evolutionary histology of the nervous system). III, (1950).</li></ol>

<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>
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<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>
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<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>
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measures of heart and ventilatory rates in freely moving crayfish

恐惧，飞行或战斗的反应作为基本研究一个有机体的下一个即将到来的食肉动物的攻击，其环境意识的生理基础。虽然目前还不知道是否无脊椎动物具有相​​同的自主神经系统脊椎动物，有证据表明无脊椎动物有一种同情的想对策，以规范的内部环境和准备的有机体行为采取行动的一个给定的刺激。此外，这种生理反应，可以切实的测量，它作为动物的内部状态的生物指数的行为。测量的生理反应，可以通过在中枢神经系统控制协调的心脑血管和呼吸系统的改变，直接关系到内部和外部的压力。更具体地说，监测心脏和通风率提供量化的应激反应并不总是行为观察措施。小龙虾是很好的模式生物，由于录制的可行性，以及小龙虾的形态丰富的历史，可以追溯到1888年，以赫胥黎，和充分研究的典型行为的心脏和换气率测量。

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

无脊椎动物展示自主同情像类似脊椎动物描述的响应。允许一个生物学指标的测量，以评估一个有机体的内部状态的心脑血管和通气系统的协调。

在自由移动的小龙虾的心脏和换气率的措施

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 次观看.  University of Kentucky. 无脊椎动物展示自主同情像类似脊椎动物描述的响应。允许一个生物学指标的测量，以评估一个有机体的内部状态的心脑血管和通气系统的协调。

视频: 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.

大鼠心脏中植入的工程组织

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

科研

生物学

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.

自由表现动物的神经回路活化的录音

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.

可视化心脏跳动果蝇</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.

小龙虾开瓶器肌肉NMJ的历史观和生理学示范

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.

电子显微镜对胚胎的制备果蝇胚胎心管

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.

电离辐射的反应定量的γH2AX灶

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.

评价以下电离辐射的空间分布的γH2AX

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.

钙火花的评估在正常肌肉纤维

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.

mRNA的衰变率的测量<em&gt;酿酒酵母</em&gt;使用<em&gt; rpb1-1</em&gt;株

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.

遗传和生物化学方法<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;蛋白齐聚评估:兰尼碱受体案例研究

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

Speed

Subtitles

Methods Article

在自由移动的小龙虾的心脏和换气率的措施