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://www.jove.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。 ）。

<ol>
	<li>Ashton, K., Wagoner, A. P., Carrillo, R., Gibson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+trait+loci+for+the+monoamine-related+traits+heart+rate+and+headless+behavior+in+Drosophila+melanogaster.">Quantitative trait loci for the monoamine-related traits heart rate and headless behavior in Drosophila melanogaster.</a> Genetics. 157, 283-294 (2001).</li><li>Azpiazu, N., Frasch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tinman+and+Bagpipe:+Two+homeo+box+genes+that+determine+cell+fates+in+the+dorsal+mesoderm+of+Drosophila.">Tinman and Bagpipe: Two homeo box genes that determine cell fates in the dorsal mesoderm of Drosophila.</a> Genes &#38; Development. 7, 1325-1340 (1993).</li><li>Badre, N. H., Martin, M. E., 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=The+physiological+and+behavioral+effects+of+carbon+dioxide+on+Drosophila+melanogaster+larvae.">The physiological and behavioral effects of carbon dioxide on Drosophila melanogaster larvae.</a> Comp Biochem Physiol A Mol Integr Physiol. 140, 363-376 (2005).</li><li>Baker, J. D., McNabb, S. L., Truman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hormonal+coordination+of+behavior+and+physiology+at+adult+ecdysis+in+Drosophila+melanogaster.">The hormonal coordination of behavior and physiology at adult ecdysis in Drosophila melanogaster.</a> J. Exp. Biol. 202, 3037-3048 (1999).</li><li>Bier, E., 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=Drosophila,+an+emerging+model+for+cardiac+disease.">Drosophila, an emerging model for cardiac disease.</a> Gene. 342, 1-11 (2004).</li><li>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=The+gene+tinman+is+required+for+specification+of+the+heart+and+visceral+muscles+in+Drosophila.">The gene tinman is required for specification of the heart and visceral muscles in Drosophila.</a> Development. 118, 719-729 (1993).</li><li>Bodmer, R., Jan, L. Y., Jan, Y. N. <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+homeobox-containing+gene,+msh-2,+is+transiently+expressed+early+during+mesoderm+formation+of+Drosophila.">A new homeobox-containing gene, msh-2, is transiently expressed early during mesoderm formation of Drosophila.</a> Development. 110, 661-669 (1990).</li><li>Cooper, A. S., 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=Monitoring+activity+of+Drosophila+larvae:+Impedance+&#38;+video+microscopy+measures.">Monitoring activity of Drosophila larvae: Impedance &#38; video microscopy measures.</a> Drosophila Information Service. 87, 85-87 (2004).</li><li>Dasari, S., 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=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. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+in+the+development+of+the+Drosophila+arista.">Cellular mechanisms in the development of the Drosophila arista.</a> Mechanisms of Development. 104, 69-78 (2001).</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=Modulation+of+Drosophila+heartbeat+by+neurotransmitters.">Modulation of Drosophila heartbeat by neurotransmitters.</a> J. Comp. Physiol. 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. Insect Physiol. 51, 489-503 (2005).</li><li>St hmer, W., Roberts, W. S., Almers, W., Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single channel recordings. The loose patch clamp, 123-132 (1983).</li><li>Wessells, R. J., 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=Screening+assays+for+heart+function+mutants+in+Drosophila.">Screening assays for heart function mutants in Drosophila.</a> Biotechniques. 37, 58-66 (2004).</li><li>Zornik, E., Paisley, K., Nichols, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+transmitters+and+a+peptide+modulate+Drosophila+heart+rate.">Neural transmitters and a peptide modulate Drosophila heart rate.</a> Peptides. 20, 45-51 (1999).</li></ol>

<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

Research

JoVE Journal

Biology

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

18.7K 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

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

在果蝇幼虫中枢神经系统的剖析

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

科研

生物学

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 ...

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

果蝇幼虫NMJ夹层

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

果蝇幼虫NMJ免疫

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

在果蝇的飞行过程中可视介导的气味追踪

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

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 ...

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 ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

幼虫，少年和成年斑马鱼的心脏解剖，斑马鱼</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

寿命测量<em&gt;果蝇</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

果蝇的制备及心功能体内使用光学相干显微镜成像纵向（OCM）

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...