生物学部，肯塔基州的年轻研究人员和本科教育尤里卡G.里布尔奖学金提供资助！办公室（肯塔基州大学）。

引言在昆虫和脊椎动物的心脏功能的细胞机制都惊人地相似 ，果蝇正在调查是在其他动物疾病（包括人类）的国家（比尔和博德默，2004年负责的基因突变 ， 。庇隆等， 2009年）。现在的各种疾病正在引起这个听话的基因生物体（东视等 ，1995;约翰逊等人，1998年; Ganetzky，2000;比尔和博德默，2004年;。Ocorr等人，2007年，B）与希望在基因治疗时间可以重新获得正常功能测试。此外果蝇作为一个好的模型在细胞水平上的生理功能。 许多过去的研究对果蝇心脏已经面向在胚胎发育方面的问题（1993年Azpiazu和Frasch，博德默1993年，博德默等 ，1990 ）。在预蛹，蛹或成人阶段（阿什顿等人 ，2001年，他和阿德勒，2001年，莫利纳和克里普斯，2001年; Ponzielli 等 ，2002; Sláma和法尔卡什，2005年发生的几个生理功能研究; Wessells和博德默，2004年）。然而，蛹期是变态和激素以及波动的各种生物胺之一。心也经历结构性转型，这可以被证明是困难的心肌细胞的生理（枣等人进行调查时，为控制变量2005年，约翰逊等人2002年，米勒，1997年; Papaefthimiou和Theophilidis ，2001年）。已知的核心是要在幼虫阶段生肌，可以很容易地被删除或留在原地 ，而受到限制，如循环激素或肽（道斯等人1995年的复合变量定义的生理盐水沐浴; 约翰逊等人1997年Dasari和库珀，2006年丰等 2004）。幼虫心脏生肌性质与哺乳动物的心脏有心脏起搏器的驱动心脏泵在流体方向洗澡机关有效的休息。 果蝇幼虫心脏变时和离子型性质的利益，因为它可以作为一个迅速的手段哺乳动物心脏功能测试的基本原则和病理的影响，以及帮助制定一个比较模式，如离子调节的细胞生理学起搏细胞。 幼虫的心是很容易在血淋巴因食物来源或动物的内在状态（Dasari和库珀，2006年，约翰逊等人1997年，2000年。尼科尔斯等1999年; Zornik等生物胺和肽1999年）。内源性或外源性化合物如何影响心脏机械的兴趣。如果我们获得了更好地了解昆虫生理学和药理学，减少对其他生物的影响可能是新颖的杀虫剂，可开发。 幼虫心脏的神经递质和调制器的作用的细胞机制尚未描述的日期尚未有足够的了解的离子流和幼虫的心，有助于调节起搏器活动的通道类型。据我们所知，没有报告，记录心肌细胞外或细胞内的录音，以评估离子电流来解决的果蝇幼虫的调制器的机械影响。 在这份报告中，我们提出各种方法来记录心率和幼虫心脏生理特性。 方法完整的幼虫： <ol><li>一个可视化的一个完整的幼虫心脏跳动的好手段是直接用显微镜;然而，幼虫仍必须保持足够的计数宫缩。我们发展的办法监测心率，在自由移动的幼虫，以及内敛的幼虫。根据实验问题上的一种方法可能比其他更合适的。 </li><li>这些方法可用于跟踪长时间，个人护理，以避免脱水。这些方法也可以用来评估在饮食上推出的药理代理人或突变线的发展，或与热休克基因的诱导研究不同时期。 </li><li>第一奔放的方法，我们称之为“蚂蚁农场”。这项技术由两片玻璃间隔一层薄薄的幼虫的食物除了板。能够可视化的​​一个重点平面内的幼虫。这种技术也被用来监测电活动，在幼虫昼夜运动（Cooper和Cooper，2004）。这项技术包括两个玻璃板（显微镜玻片上，75 x 25毫米; J.梅尔文释放的品牌），狭义的间隔（1对1。5毫米）除了一层薄薄的幼虫的食物（如潮湿的玉米粉，刘易斯，1960年修改后的版本），这样的幼虫能够可视化的​​一个重点平面内。凝胶电泳板常用的垫片（迷你凝胶Bio - Rad公司;生命科学研究，大力士，CA 94547，美国）很好地工作，因为它们可以购买不同厚度为1，第2或第3龄幼虫的蚂蚁农场程序使用。也是一种选择是使用一个给定的厚度的固体塑料和切出该地区使用的爬行空间。 </li><li>为了防止从两个玻璃的板所需厚度的塑料用于边缘爬出来的幼虫。稍微倾斜的平台，在20至45度，导致幼虫保持，大部分时间，他们的头部，指出向下，尾部含有上述食品或在食品层内的空气通过的气孔。 </li><li>这种“蚂蚁农场技术”还允许与厚度均匀的单一食品平面内的视频影像。在此配置中的幼虫往往不抓取整个食品，而是吃，并逐步在二维平面上左右移动。预计从镜显微镜阶段底部的白色光，以便它可以被移到相应心脏或两个气管而心脏收缩移动最好的对比度。显微镜（可调变焦0.67至4.5;世界精密仪器;型号501379）。一个2X的基本目标和管目标0.5X是用来获得足够的空间分辨率和放大倍率，覆盖0.5厘米的矩形，一个1cm。使用一个安装摄像头是通过一个三目挂载（敏通，MTV;世界精密仪器）。环境温度保持在20 ° C。 <img src="/files/ftp_upload/1596/1596fig1.jpg" alt="图1" /> 图1。一个完整的第三龄幼虫的背视图。气管的运动，由于拉动发自内心的附件是用来观察心率。 </li><li>要监视自由活动的动物，可以将几滴稀的食物混合比动物头。这项工作应在一个玻璃盘，使透射光可以通过可视化心管的幼虫。设立上文所述相同的显微镜可用于观看心脏跳动，并取得计数。 </li><li>另一种方法是限制使用双节棍在玻片上的磁带和配售幼虫腹磁带（Baker等，1999），幼虫到一个位置。但是这种方法并不能很好的工作，如果磁带弄湿喂养幼虫。为了避免弄湿之一的磁带，可以使用凡士林（小针注射围绕基地的幼虫和周围的磁带边缘）。在这里，人们可以随着时间的推移，而不必追成焦点平面的幼虫或虽然这是一盘移动幼虫饲料。如果一个人希望自由幼虫的磁带，可以湿润，失去它的附着力动物。 </li></ol>如果没有兴趣释放幼虫的实验，人们可以使用的永久的方法抑制动物的幼虫通过粘合载玻片。用超级胶水方法，动物可以吃，甚至可以在潮湿的解决方案覆盖，而其余坚持以玻璃盖玻片。的程序是： <ol><li>取一干净的幻灯片，并将其放置在它的一端盖玻片。 </li><li>小民建联的强力胶把盖玻片的一个角落。 </li><li>找到您的果蝇幼虫，从试管中取出。 </li><li>放置在培养皿中的幼虫和冲洗一个少量的水，以消除任何多余的食物。 </li><li>沉浸扩孔用一个小的组织或纸巾的一角食物。 </li><li>用镊子轻轻地拿起幼虫和它从您的盖玻片上，另一端放置在幻灯片上。 </li><li>放置在显微镜下的幻灯片，并调整您的贷款幼虫。幼虫应在其背面朝上的胃。您可以分清双方的幼虫，因为他们的备份功能“赛车条纹”，这是气管。胃有淡淡的横向凹槽，沿着它的运行很细的黑毛。 </li><li>如果幼虫正面临着不正确的方法，只需打开您的镊子轻轻翻转它的正确途径。 </li><li>有了一个新的镊子一套专门用于胶水从在你的封面年底下降一个小民建联滑，并放置在另一端的角落。您应该使用的胶水小幅量;足以涵盖镊子头。另外，确保你擦去你的镊子的两端，使他们不成为​​粘关闭。 </li><li>在显微镜下，仔细检查，以确保幼虫仍然是在正确的位置。如果它翻了过来，见第八步。 </li><li>现在，用于处理幼虫镊子，拿起幼虫把它轻轻地放在新鲜的胶修补。确保黑嘴挂钩靠近或在盖玻片边缘，既不他们或褐色气孔与胶水接触。 </li><li>小心按下幼虫向下压平。 </li><li>现在，幼虫在地方，你可以管理要测试他们的物质。 </li><li>这是最好的，如果你使用注射器制订的实质和幼虫的头部在小剂量的摄取。 </li><li>最后，心率计数脉冲的气孔在一分钟内可以观察到。 </li></ol> 在原地 ：解剖和计数心率： 筹备工作是缝沿中腹的纵轴和牵制持平。准备菜包括玻片（VWR）磁带（百思买零售出来，让）坚持一方。在磁条中心孔允许准备与透射光观察。解剖针（精细的科学工具，WA）是弯曲和粘回形针。回形针很容易被操纵的磁带上，举行的地方鱼片的准备。这种类型录音盘先前已利用寄希望于从水蛭腹神经索（Muller 等 ，1981）分离出神经节。 <ol><li>完整的第三龄幼虫放在夹层板。腹侧旋转的幼虫，直到气管不再通过角质层可见。 </li><li>将四个完整的幼虫引脚。有两个引脚口钩两侧，两个引脚上的气孔两侧。 </li><li>加入生理盐水，做一个横向切开一个小的距离头管脚，然后继续向下纵轴长的水平切口。 </li><li>从真正的心脏停止切割短距离。割开真实的心和沿一侧的气孔。 </li><li>抬起一条背针和钩下方角质层。使用PIN打开体腔和传播打开它。其余引脚重复。 </li><li>取出内脏，注意不要损伤气管或心脏。应该指出，一些建筑物被附加到心，如果去掉可能会损坏它。这些措施包括几个脂肪体，脑。如果你可以看到被删除的胆量，而拉长的心，立即停止试图删除该结构。 </li></ol>现在的核心是暴露和暴露实验条件的准备。 <img src="/files/ftp_upload/1596/1596fig2.jpg" alt="图2" /> 图2，3路龄的腹夹层直接查看心。动物在它的后面钉解剖后用来直接适用于中枢神经系统或不完整的，对心脏的化合物。小箭头表示心脏和主动脉横断背血管研究（见下文）分隔。 TR，气管; SP，气孔，H心; AO，主动脉。 这夹层技术已被用于直接评估对果蝇幼虫的心脏（谷和辛格，1995年）的药理剂。清扫时间为3-6分钟。放松，而在HL3解剖后的3-5分钟的盐水沐浴的准备工作。 HL3生理盐水进行了仔细的控制，在pH值7.2，因为HR减慢在高pH值和速度在较低的pH值（Badre等，2005 ）。顾和Singh（1995）用于心脏的药理分析pH值7.0，也表明保持生存能力。在这些试验中，生理盐水保持曝气搅拌的解决方案，通过反复注射生理盐水，通过21号针，到一个烧杯中。 要量化人力资源可用于直接观察或图像可以被记录在VHS录像带，或在屏幕上进行播放的数码相机。一个光电二极管的方法以前曾描述（Dasari和库珀，2006）。 暴露在热休克： <ol><li>解剖幼虫。 </li><li>广场解剖盖板上的准备。 </li><li>热水浴。 </li><li>检查浴缸内浮动盖板上一个温度探测器温度。 </li><li>当温度是令人满意的，在洗澡的地方编制和覆盖板。 </li><li>留在编制所需的时间量，并在完成时删除。 </li></ol>人们可以利用其试验所需的每一个热脉冲和温度。 心电驱动的步伐： <ol><li>填补一个协调中心电极（直径约20微米），使用注射器插入开口端密封，并绘制在室温（21℃），飞入生理盐水（HL3）电极。的机械臂安装到电极，检查日发送线是在生理盐水内的电极和刺激是关闭的。 </li><li>放置在解剖镜下一个果蝇幼虫3龄幼虫心脏解剖的准备，并确保到位。放入生理盐水的准备，谨慎地避免接触的准备与引脚和双方的接地线。 </li><li>使用机械手到心脏的顶部电极尖端位置，需要调整显微镜的焦点。实现这一目标的最佳方式是电极上的重点，同时针尖向下移动。 </li><li>当针尖被认为是在位置，应该打开的刺激（型号S9草仪器，美国，马萨诸塞州，昆西）在2赫兹频率，持续时间在0.5到1毫秒的电压的最低设置。延迟设置应介于12和14。 </li><li>同时观察心脏的节奏，频率应转向约3 Hz的电压缓慢上升，直到看到心脏的节奏对应的刺激所产生的节奏。解剖飞心跳通常在大约2赫兹的频率，所以当刺激的节奏高于内禀增长率将会看到，心​​，心会被视为击败更快的速度比正常。电压应不超过约20个细胞的损伤，可发生在高电压，准备无用V。通常情况下，可以刺激2和1.8 V之间的心，这取决于距离和对心脏创建的印章。 </li><li>如果达到最大电压，被认为是没有效果，可以逆转的极性。如果这也不起作用时，探头应提出更好地树立对心脏的连接。 </li><li>一旦建立一个刺激驱动的节奏，节奏可以操纵不同的频率和持续时间设置。应该知道，不过，4赫兹以上的频率可以把手足抽搐的心。可以添加各种醇类及药理制剂和缝隙连接的影响，可以观察到，一旦建立所需的节奏。封锁的缝隙连接不进行电流和传播的节奏将停止。 <img src="/files/ftp_upload/1596/1596fig3.jpg" alt="图3"/> <img border="0" src="/files/ftp_upload/1596/1596fig4tmb.jpg" alt="图4"/>看到这个数字更大的版本， <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/1596/1596fig4.jpg">请点击</a>这里。 </li></ol>测量场电位： <ol><li>玻璃电极是由玻璃Kimax（外径：1.5毫米）。玻璃管拉火抛光生产内径从10到20微米的补丁提示。 </li><li>电极内腔充满了沐浴液。放大器和线到电脑上的记录是用于细胞内的录音低于相同。 </li><li>管腔一个“宏观补丁”的记录电极（Stühmer等 ，1983）是直接放在心脏地区。自发的肌纤维起搏产生一个领域的潜力，可与中心电极监测。 </li><li>需要注意的是轻轻地降低流明，提高它在每个心要监视的地区的。通过宏观补丁电极记录的潜力。 </li></ol>直接计数心率和电气事件监测是可能的。改变洗浴的媒体，而录音这些潜力，也是可行的驴场电位的影响。 测量细胞内的潜力： 要监视的一个尖锐的细胞内电极（20至30毫欧电阻）与3米氯化钾填补刺穿心脏地区的心肌细胞跨膜电位。可以使用一个标准的头阶段，细胞内记录放大器;然而，我们采用的一种模式Axonclamp 2B（分子器件，美国加利福尼亚州的桑尼维尔市，美国）放大器和1个LU头阶段。看来，心的尾部到底是更严格的，这是起搏细胞自收缩在本地区启动的时间（Badre等 ，2005年的大部分; 2006年Dasari和库珀; Dasari等人，2007年。 ）。

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+influence+of+serotonin+on+the+larval+heart+of+Drosophila+melanogaster.">Direct influence of serotonin on the larval heart of Drosophila melanogaster.</a> J. Comp. Physiol. B. 176, 349-357 (2006).</li><li>Dasari, S., Viele, K., Turner, A. C., 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=Influence+of+p-CPA+and+MDMA+on+physiology,+development+and+behavior+in+Drosophila+melanogaster.">Influence of p-CPA and MDMA on physiology, development and behavior in Drosophila melanogaster.</a> European J. Neurosci. 26, 424-438 (2007).</li><li>Dowse, H., Ringo, J., Power, J., Johnson, E., Kinney, K., White, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+congenital+heart+defect+in+Drosophila+caused+by+an+action-potential+mutation.">A congenital heart defect in Drosophila caused by an action-potential mutation.</a> J. Neurogenetics. 10, 153-168 (1995).</li><li>Dulcis, D., Levine, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamatergic+innervation+of+the+heart+initiates+retrograde+contractions+in+adult+Drosophila+melanogaster.">Glutamatergic innervation of the heart initiates retrograde contractions in adult Drosophila melanogaster.</a> J. Neurosci. 25, 271-280 (2005).</li><li>Feng, Y., Ueda, A., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+minimal+hemolymph-like+solution,+HL3.1,+for+physiological+recordings+at+the+neuromuscular+junctions+of+normal+and+mutant+Drosophila+larvae.">A modified minimal hemolymph-like solution, HL3.1, for physiological recordings at the neuromuscular junctions of normal and mutant Drosophila larvae.</a> J. Neurogenetics. 18, 377-402 (2004).</li><li>Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+ion+channel+dysfunction+in+Drosophila.">Genetic analysis of ion channel dysfunction in Drosophila.</a> Kidney International. 3, 766-771 (2000).</li><li>GG, G. u. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+analysis+of+heartbeat+in.">Pharmacological analysis of heartbeat in.</a> Drosophila. J. Neurobiol. 28, 269-280 (1995).</li><li>He, B., Adler, P. 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B. 167, 89-97 (1997).</li><li>Johnson, E., Ringo, J., Bray, N., Dowse, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+pharmacological+identification+of+ion+channels+central+to+the+Drosophila+cardiac+pacemaker.">Genetic and pharmacological identification of ion channels central to the Drosophila cardiac pacemaker.</a> J. Neurogenetics. 12, 1-24 (1998).</li><li>Johnson, E., Ringo, J., Dowse, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+and+heterologous+neuropeptides+are+cardioactive+in+Drosophila+melanogaster.">Native and heterologous neuropeptides are cardioactive in Drosophila melanogaster.</a> J. Insect Physiol. 1, 1229-1236 (2000).</li><li>Johnson, E., Sherry, T., Ringo, J., Dowse, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+the+cardiac+pacemaker+of+Drosophila:+cellular+mechanisms.">Modulation of the cardiac pacemaker of Drosophila: cellular mechanisms.</a> J. Comp. Physiol. B, Biochem., Systemic, Environmental Physiol. 172, 227-236 (2002).</li><li>Lewis, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+standard+food+medium.">A new standard food medium.</a> Drosophila Inform. Serv. 34, 117-117 (1960).</li><li>Miller, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+circulation+in+insects.">Control of circulation in insects.</a> General Pharmacol. 29, 23-38 (1997).</li><li>Molina, M. R., Cripps, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ostia,+the+inflow+tracts+of+the+Drosophila+heart,+develop+from+a+genetically+distinct+subset+of+cardial+cells.">Ostia, the inflow tracts of the Drosophila heart, develop from a genetically distinct subset of cardial cells.</a> Mechanisms of Development. 118, 51-59 (2001).</li><li>Muller, K. J., Nicholls, J. G., Stent, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neurobiology of the Leech. , 254-254 (1981).</li><li>Nichols, R., Kaminski, S., Walling, E., Zornik, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulating+the+activity+of+a+cardioacceleratory+peptide.">Regulating the activity of a cardioacceleratory peptide.</a> Peptides. 20, 1153-1158 (1999).</li><li>Ocorr, K., Akasaka, T., Bodmer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+cardiac+disease+model+of+Drosophila.">Age-related cardiac disease model of Drosophila.</a> Mechanisms of Ageing and Development. 128, 112-116 (2007a).</li><li>Ocorr, K. A., Crawley, T., Gibson, G., Bodmer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variation+for+cardiac+dysfunction+in+Drosophila.">Genetic variation for cardiac dysfunction in Drosophila.</a> PLoS ONE. 2, e601-e601 (2007).</li><li>Papaefthmiou, C., Theophilidis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+method+for+recording+the+electrical+activity+of+the+isolated+heart+of+the+adult+Drosophila+melanogaster.">An in vitro method for recording the electrical activity of the isolated heart of the adult Drosophila melanogaster.</a> In Vitro Cellular &#38; Developmental Biol. Animal. 37, 445-449 (2001).</li><li>Peron, S., Zordan, M. A., Magnabosco, A., Reggiani, C., Megighian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+action+potential+to+contraction:+Neural+control+and+excitation-contraction+coupling+in+larval+muscles+of+Drosophila.">From action potential to contraction: Neural control and excitation-contraction coupling in larval muscles of Drosophila.</a> Comp. Biochem.Physiol. A: Molecular &#38; Integrative Physiol. 154, 173-183 (2009).</li><li>Ponzielli, R., Astier, M., Chartier, A., Gallet, A., Therond, P., Semeriva, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+tube+patterning+in+Drosophila+requires+integration+of+axial+and+segmental+information+provided+by+the+Bithorax+Complex+genes+and+hedgehog+signaling.">Heart tube patterning in Drosophila requires integration of axial and segmental information provided by the Bithorax Complex genes and hedgehog signaling.</a> Develop. 129, 4509-4521 (2002).</li><li>Sl ma, K., Farkas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heartbeat+patterns+during+the+postembryonic+development+of+Drosophila+melanogaster.">Heartbeat patterns during the postembryonic development of Drosophila melanogaster.</a> J. 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<ol><li>解剖工具：精细＃5镊子和罚款剪刀（获得精细科学的工具（美国）公司，373 - G的复古公园径，福斯特市，CA 94404-1139） </li><li>解剖镜下解剖和计数心率与变焦功能。这是心的焦点刺激也同样需要。 </li><li>一个正直的目标（4倍和20倍）的复合式显微镜是对细胞外和细胞内的录音使用。 标准细胞内的放大器和A / D板线录制到计算机。电信号记录上线到PowerLab/4s接口（澳大利亚ADInstruments，）。我们使用标准软件名为图表或范围ADInstruments。 </li><li>化学品：所有生理盐水的化学品是从Sigma公司（圣路易斯，密苏里州）获得。 </li><li>细胞内的录音，我们使用的玻璃毛细管（金控，不伦瑞克，ME，04011，USA的目录＃30-31-0）和焦距macropatch记录和刺激电极，我们使用Kimax - 51，金布尔产品艺术。 34502号，内径0.81.1毫米，长度100mm。细胞内的电极应该有一个20至30毫欧电阻。 macropatch电极构造掰后的精尖是从一个电极拉马的玻璃的一角。折断提示需要一个直径约20μM清洁垂直打破。笔尖再热抛光，内径10μm左右的焦距外记录。对于刺激电极驱动的心，我们用一个直径约20-50微米的最后一角。电极轴是运行在一个加热元件，以使其一个渐进的弯曲约45度弯曲。这将产生一个平坦的心管或垂直电极的微型机械臂的角度流明，会产生约45度的准备。 </li></ol>

monitoring heart function in larval drosophila melanogaster for physiological studies

我们目前各种方法记录在幼虫心脏功能<em&gt;果蝇</em&gt;。该方法允许在奔放和内敛的整个幼虫测量心率。对于心脏周围环境的直接控制的另一种方法采用解剖的幼虫和去除内脏器官，以洗澡所需的化合物的心。暴露的心也使膜电位监测，可以由心肌细胞和沿心管导电产生的离子电流的洞察力。这些方法有不同的优势和劣势，讨论了今后的实验。幼虫的心准备提供了一个额外的模型除了<em&gt;果蝇</em&gt;骨骼NMJ探讨细胞功能的细胞内钙离子调控的作用。学习有关的基本形状在不同物种的心肌细胞的动作电位的离子电流的，人们可以希望得到一个负责在哺乳动物身上的各种疾病的特定基因相关联的已知离子功能障碍处理。

Watch this Scientific Journal Video about Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies at JoVE.com

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

我们目前各种方法来监测心脏功能，在幼虫<em&gt;果蝇</em评估与处理的缝隙连接，离子通道基因突变，调节心脏起搏器活动和药理研究功能的问题。

在幼虫的监测心脏功能果蝇生理研究

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

monitoring-heart-function-larval-drosophila-melanogaster-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

18.8K 次观看.  University of Kentucky, Lexington. 我们目前各种方法来监测心脏功能，在幼虫果蝇

视频: Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

高分辨率视频跟踪移动的，在成年果蝇。

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

科研

生物学

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

荧光标记的果蝇心结构

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

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 ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

半自动小红桃光学心跳分析

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Novel And Efficient Method for Drosophila Heart Fluorescence Staining with Cryosectioning

Drosophila heart models are widely employed in studying cardiac aging and modeling&#160;human cardiac diseases. However, the dissection of Drosophila hearts before imaging is a meticulous time-intensive process that requires advanced training and motor skills. To address these challenges, we present an innovative protocol that utilizes cryosectioning for the fluorescence imaging of Drosophila heart tissue. The protocol has been demonstrated in imaging the adult Drosophila heart but could be adapted for developmental stages. The method enhances both the efficiency and accessibility of fluorescence staining while preserving the integrity of the tissue. This protocol simplifies the process without compromising the quality of imaging, thereby reducing the dependency on technicians with highly developed training and motor skills. Specifically, we replace complex techniques, such as capillary vacuum suction, with more straightforward methods like tissue embedding. This approach allows for the visualization of cardiac structures with greater ease and reproducibility. We demonstrate the utility of this protocol by effectively detecting key cardiac markers and&#160;achieving high-resolution fluorescence and immunostaining imaging that unveils intricate details of heart morphology and cellular organization. This method provides a robust and accessible tool for researchers exploring Drosophila cardiac biology, facilitating detailed analyses of heart development, function, and disease models.

The Drosophila heart sectioning and fluorescence imaging protocol simplifies studying heart structure and pathologies. This approach involves straightforward sectioning, staining, and imaging, bypassing the technical expertise needed for traditional dissection. It enhances accessibility, making Drosophila a more widely usable model for cardiac-related research within the broader scientific community.

使用冷冻切片进行 果蝇 心脏荧光染色的新型高效方法

Drosophila heart models are widely employed in studying cardiac aging and modeling&#160;human cardiac diseases. However, the dissection of Drosophila ...

遗传学

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

在自发活动的测定<em&gt;果蝇</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

神经科学

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

一种测定果蝇运动活性的简单技术

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

行为学

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

第三星果蝇中枢神经系统活动的电生理记录

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...

Developing Drosophila melanogaster Models for Imaging and Optogenetic Control of Cardiac Function

Using Drosophila melanogaster (fruit fly) as a model organism has ensured significant progress in many areas of biological science, from cellular organization and genomic investigations to behavioral studies. Due to the accumulated scientific knowledge, in recent years, Drosophila was brought to the field of modeling human diseases, including heart disorders. The presented work describes the experimental system for monitoring and manipulating the heart function in the context of a whole live organism using red light (617 nm) and without invasive procedures. Control over the heart was achieved using optogenetic tools. Optogenetics combines the expression of light-sensitive transgenic opsins and their optical activation to regulate the biological tissue of interest. In this work, a custom integrated optical coherence tomography (OCT) imaging and optogenetic stimulation system was used to visualize and modulate the functioning D. melanogaster heart at the 3rd instar larval and early pupal developmental stages. The UAS/GAL4 dual genetic system was employed to express halorhodopsin (eNpHR2.0) and red-shifted channelrhodopsin (ReaChR), specifically in the fly heart. Details on preparing D. melanogaster for live OCT imaging and optogenetic pacing are provided. A lab-developed integration software processed the imaging data to create visual presentations and quantitative characteristics of Drosophila heart function. The results demonstrate the feasibility of initiating cardiac arrest and bradycardia caused by eNpHR2.0 activation and performing heart pacing upon ReaChR activation.

Developing Drosophila melanogaster Models for Imaging and Optogenetic Control of Cardiac Function

The present protocol describes the generation of Drosophila melanogaster expressing eNpHR2.0 or ReaChR opsins in the heart for OCT imaging and optogenetic heart pacing. Detailed instructions for Drosophila OCT imaging and heart beating modulation, including the simulation of restorable heart arrest, bradycardia, and tachycardia in live animals at different developmental stages, are reported.

开发用于心脏功能成像和光遗传学控制的 黑腹果蝇 模型

Using Drosophila melanogaster (fruit fly) as a model organism has ensured significant progress in many areas of biological science, from cellular ...

生物工程

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

幼虫活动的测量<em&gt;果蝇</em&gt;活动监视器

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

在幼虫的监测心脏功能果蝇生理研究

摘要

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此视频中的章节

高分辨率视频跟踪移动的，在成年果蝇。

荧光标记的果蝇心结构

可视化心脏跳动果蝇

半自动小红桃光学心跳分析

使用冷冻切片进行果蝇心脏荧光染色的新型高效方法

在自发活动的测定

一种测定果蝇运动活性的简单技术

第三星果蝇中枢神经系统活动的电生理记录

开发用于心脏功能成像和光遗传学控制的黑腹果蝇模型

幼虫活动的测量

在幼虫的监测心脏功能果蝇生理研究

摘要

探索更多视频

此视频中的章节

高分辨率视频跟踪移动的，在成年果蝇。

荧光标记的果蝇心结构

可视化心脏跳动果蝇

半自动小红桃光学心跳分析

使用冷冻切片进行 果蝇 心脏荧光染色的新型高效方法

在自发活动的测定

一种测定果蝇运动活性的简单技术

第三星果蝇中枢神经系统活动的电生理记录

开发用于心脏功能成像和光遗传学控制的 黑腹果蝇 模型

幼虫活动的测量

使用冷冻切片进行果蝇心脏荧光染色的新型高效方法

开发用于心脏功能成像和光遗传学控制的黑腹果蝇模型