Name: imaging calcium in drosophila at egg activation
Uploaded: 2016-08-06T14:00:00.000+00:00
Duration: 7 min 45 s
Description: Watch this Scientific Journal Video about Imaging Calcium in Drosophila at Egg Activation at JoVE.com

我们感谢劳拉班普顿，亚历克斯·戴维森，理查德·帕顿，阿查里雅田对这个手稿的准备过程中提供援助;马里亚纳Wolfner对鸡蛋激活讨论;马特·韦兰成像的支持;和胡楠在实验室普遍支持。 这项工作是由剑桥，ISSF的大学TTW [授权号097814]的支持。

注:在常温下进行所有步骤，除非另有说明。 1.准备解剖注意:这里描述的步骤都是按照E. Gavis，普林斯顿大学和威尔等。人 18。前成像执行以下步骤两天。 <ol><li>取含有大约10毫升固体食物蝇小瓶中并干燥酵母的少量添加到一个角，小于1g是足够的。 注:有关飞小瓶或食品信息，请参阅" 果蝇维护"19。 </li><li>加入1 - 水3滴，以水化酵母。 </li><li>预留小瓶在45°角，并允许3 - 为对酵母5分钟以占用水。 </li><li>而酵母是吸收水，选择一个瓶表达由浴盆-VP16GAL4驱动UASt- MYR GCaMP5 20蝇（简称GCaMP5），该最近出现（一myristoy被选择的GCaMP5的迟来形式通过在蛋拴系的GECI向膜），以提供高的信噪比。 </li><li>麻醉苍蝇在瓶的 CO 2气体。一定要保持瓶口倒置，以免失去苍蝇在湿的食物。 </li><li>尖端麻醉苍蝇到CO 2垫和排序10 - 15名女性和男性5使用解剖显微镜下画笔。注:这是标准的，包括男性为解剖提供健康的女性。 </li><li>当来自步骤1.3小瓶准备就绪时，所选择的苍蝇添加到小瓶中。注意保持小瓶水平，直到苍蝇变得活跃。 </li><li>放置在25℃的小瓶约36小时。 </li><li>从CO 2垫到瓶子或丢弃返回剩余的苍蝇。 </li></ol> 2.解剖果蝇卵巢注意:这里描述的步骤都是按照威尔等。人。 <s达&gt; 18。 <ol><li> 36小时后，从CO 2 yeasted小瓶麻醉苍蝇。 </li><li>一旦小瓶内部的苍蝇麻醉提示他们到CO 2的垫用解剖显微镜下漆刷进行排序。 </li><li>选择一个女性。 </li><li>隔离从阴两个卵巢。有关完整详细的协议看威尔等。人 18。综上所述: <ol><li>将含氧卤烃油（系列95）的一小滴在1.5号，22×40mm的盖玻片。 </li><li>把握用镊子阴在腹部的前部。 </li><li>在解剖显微镜下握住女性，使用第二套钳轻轻地去除女性的后路。 </li><li>擦去从第二钳后组织。 </li><li>挤在与由前腹部的后部的第2钳子腹部空腔。这两个不透明卵巢应坚持镊子时，外面的联邦紧急事务管理局勒。 </li><li>触摸卵巢进油。 </li><li>擦去镊子除去从钳子的任何组织。 </li><li>从一飞获得3盖玻片，每个包含卵巢 - 重复上述步骤（2.4.7 2.4.1）。 </li></ol></li></ol> 3.隔离单个成熟卵子<ol><li>根据与来自步骤2.4设置为4X，在第一盖玻片的倍率的标准解剖显微镜，通过撕开输卵管分开的两个卵巢。 </li><li>介绍一对封闭钳成一个卵巢的中心，注意不要刺破任何蛋室。 </li><li>插入一个标准的解剖探入卵巢的中心封闭镊子旁边。 </li><li>通过朝后极，其中，成熟的卵子（阶段14蛋室）位于卵巢轻轻拖曳解剖探针。 </li><li>重复步骤3.1 - 取决于卵巢和蛋的数量的大小的5倍 - 3.4 2。 注:CO 2的添加</子&gt;通常会导致将出现与凸起背侧附属物比预沉积卵较大的单个蛋的沉积。因为它已经被阴内激活这个蛋应该丢弃成像。 注:成熟的卵子有可能轻松地从卵巢结缔组织分隔。 </li><li>如果没有成熟的卵子都是卵巢的外部可见，继续轻轻地用解剖探头戏弄。 </li><li>在盖玻片的中心的线5 - 一旦成熟卵是对卵巢的外盖玻片可见，操纵3。 注:为达到最佳效果，沿鸡蛋的一侧轻轻地运行探头。 注意:不要拉背附肢，否则会损坏卵子。 注意:根据未来成像设置，对准鸡蛋约200微米相距垂直于盖玻片最大成像区。 </li><li>一旦对准，通过使用f起对准鸡蛋拖动距离取出卵巢组织的其余部分orceps。 </li><li>通过与医疗擦拭涂抹角落删除多余的油。小心不要碰到成熟的卵子与医疗擦拭那些接触将失去作为成熟的卵子。 </li><li>上绘制盖玻片的十字准线与一个标记，以简化定位在显微镜下的样品。 </li><li>坐落在一个安全的地方盖玻片，离开盖玻片上的准备鸡蛋室休息5 - 10分钟。这允许蛋在油定居。样品可以使用到1小时后淋巴结清扫术。 </li><li>重复步骤3.1 - 3.11盖玻片的其余部分与他们的卵巢。 </li></ol> 4.准备成像<ol><li>带来制备活化缓冲器15至室温。 注意:激活缓冲液是低渗缓冲液:3.3毫的NaH 2 PO 4，16.6毫KH 2 PO 4，10 mM氯化钠，50mM的氯化钾，5％的聚乙二醇8000，2毫氯化钙2，调至pH 6.4以1: 5比氢氧化钠:KOH。有关详细信息，请参阅马霍瓦尔德，1983 15。活化缓冲液的等分可以储存在-20℃下个月，并在4℃下最多2周。 </li><li>精心准备运盖玻片，活化缓冲液和玻璃吸管的成像设备。 注意:宽视场，共聚焦，旋转光盘或光片显微镜都可以使用。我们建议使用倒置显微镜。利用共聚焦显微镜获得了我们的代表性的结果。 </li><li>适合成像整个成熟的卵子一个目标应（这里:20X 0.7 NA）选择。 </li><li>安装在显微镜阶段的第一个准备盖玻片。小心不要打破舞台上的盖玻片。 </li><li>定性选择的视场与成熟卵室进行激活。不要刺破形象蛋室或那些在细胞膜起泡。 注意:对于步骤4.6 - 4.7，软件命令将视显微镜和制造商的不同而不同。 </li><li>组影像对位米标准GFP激发（488纳米）和发射（500 - 580纳米）。还成立了以可视化和定向的成熟卵子和流离失所油明视场视图。 </li><li>选择最低的Z平面，其中成熟卵是可见的，并设置一个完整的Z堆栈应采取的40微米每步约2微米。所以Z堆栈收购之间没有延迟设置参数。设置的时间序列来捕获约30分钟。 </li></ol> 5.成像离体激活蛋<ol><li>启动图像捕捉。 </li><li>等待1 - 要获得2个全Z-栈。这说明之前激活成熟的卵子。 </li><li>撤回大约300微升活化缓冲剂成玻璃吸管。 </li><li>定位含有活化缓冲液中在45°角盖玻片上方的玻璃吸管的前端，慢慢地吸移管移入光束路径直到它直接上面的成熟卵被成像。玻璃吸管应为1 - 2厘米油上方。 </li><li>从玻璃吸管活化缓冲液2滴，在不到5秒 - 加入1。这应该置换的油。避免加入过量的活化缓冲或触摸与玻璃吸管蛋，因为它可以导致成熟卵子的位移。小心不要碰到盖玻片用吸管，因为这可能会导致在焦平面或气泡的变化，形成了客观和盖玻片之间的浸油。 </li><li>虽然图像采集过程中添加活化缓冲液，检查亮场通道，确保油已被激活缓冲流离失所。 注意:这将首先显示为阴影，由于移液管断裂的光束路径，但也将导致在小油滴的外观。有些油滴可以与成熟的卵子，这将影响实验结果和图像质量保持联系。 </li><li>如果油没有从初始加成活化缓冲液重复的位移步骤5.4 - 50.6。 </li><li>一旦油被成功移动，使时间序列直到完成运行。 注:由于在激活卵母细胞肿胀，有的蛋商会将显着增加时激活缓冲区移出焦平面或视场。在这种情况下，停止采集并安装下一个盖玻片。 </li><li>图像序列已完成收购后，保存数据。 </li></ol> 6.收购后图像处理<ol><li>打开使用斐济（http://fiji.sc/Downloads#Fiji），或同等图像处理软件中的数据集，并选择相关文件进行分析。 </li><li>要建立采集数据的投影选择:图像&gt;栈&gt; Z-项目。 </li><li>选择开始的Z-切片和停止的Z-片，典型地整个部分。选择投影式，通常设置为"最大强度"。选中复选框"所有时间框架"来选定的设置适用于整个系列。 </li><li>如果一个以上的荧光信道已被成像，使用该频道工具来启用颜色之间的运动，使得复合材料，或者使图像的灰度。选择图像&gt;颜色&gt;频道工具。 </li><li>要调整图像亮度和对比度选择图像&gt;调整&gt;亮度/对比度。 </li><li>要导出单个图像，移动滑块时间表的有关框架，然后选择文件&gt;另存为&gt; JPEG，或任何首选格式。 </li><li>选择导出的文件的位置和名称，然后保存。 注意从成像软件的帧频将被要求提供在图像上的时间戳。 </li><li>以时间序列导出为影片选择:文件&gt;另存为&gt; AVI，然后选择帧速率（〜每秒7帧）。 </li><li>选择导出的文件的位置和名称，然后保存。 </li><li>为了节省在斐济格式调整文件，选择File&gt;保存。 </li></ol>

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The first critical step in this protocol is isolating the mature eggs without damage. This can be achieved by gentile maneuvering of the eggs with the dissecting probe. Practice will enable this manipulation to be executed without damage to the eggs. A second crucial step is avoiding the loss of the sample when activation buffer is applied to the mature egg in oil. Application of the activation buffer should be done slowly and without contact with the coverslip. This step can be challenging if the microscope set-up does not allow for an easy access to the sample. Moving axillary parts of the microscope, such as a temperature incubator, is advisable. 
Modifications to this protocol can be made in order to visualize other fluorescently tagged factors, instead of Ca2+ at egg activation. More generally, different stages of Drosophila development or the results of adding a different buffer could be analyzed using this protocol. 
Troubleshooting may be required if the settings on the microscope are not optimal for visualizing the signal from the sample. This can be achieved by increasing or decreasing the laser power, altering capture range and adjusting the Z-stack parameters. Another issue might be eggs consistently moving out of the field of view upon the addition of activation buffer. If this happens regularly, allow the mature eggs to settle on the coverslips for a longer period of time, 15 to 20 minutes. Beware that using a higher viscosity oil will alter the displacement of the oil by activation buffer. 
This technique presented here is limited by the working distance of the objective, dehydration of the mature eggs and the ability to dissect the mature eggs without damage. However, when compared to in vivo imaging methods where the mature oocyte passes through the adult female and is deposited 13, our method enables more spatial resolution, the option to physically manipulate the mature egg before activation and the ability to test the role of egg activation without fertilization. 
There are many potential future applications of this technique, including testing reporter constructs for cellular components at egg activation and visualizing the Ca2+ wave in mutant backgrounds 14,24. Together with experimental and genetic tools, the ex vivo egg activation assay presented here enables the study of the trigger, propagation and downstream effects of the universally conserved Ca2+ wave.

在蛋（卵母细胞）的激活细胞内钙的变化2+浓度是所有研究的有机体1,2的保守成分。该事件启动了广泛的Ca 2+依赖性的过程，包括细胞周期的恢复和存储mRNA的翻译。由于这一要求的钙离子 ，可视化细胞内钙离子浓度瞬时变化是关键的兴趣。 历史上，被选定为基于它们的卵的大小和可用性上激活卵子的研究模式生物。各种可视化的方法已被用于跟踪和细胞内Ca量化改变在这些系统中2+浓度，其中包括:在青鳉鱼3的光蛋白水母发光蛋白; 的 Ca 2+敏感的荧光染料如的Fura-2在海胆和仓鼠4,5;和钙绿-1-葡聚糖在爪 6。的产生和基因编码的钙指标（GECIS）改进已经改变可视化钙动力学体内 7的能力。这些遗传构建体在特定组织中表达，并限制对侵入组织标本8的必要性。 GCaMPs是绿色荧光蛋白（GFP）基类已经非常有效GECIS的，由于其高的 Ca 2+亲和力，信噪比和能力来定制9-11。在钙离子的存在下，在GCaMP复杂经历一系列的构象变化，开始与钙离子的钙调蛋白的结合，即结果在GFP元器件9的增加的荧光强度。 GCaMPs已在研究被广泛用在果蝇神经元的可视化细胞内钙12的变化。最近applicati在GCaMP技术成熟果蝇卵可视化钙离子已经揭示了蛋激活13,14一个短暂的钙波。在钙离子波可以在低倍镜下采用体外活化试验13,14 体内排卵13期间或在更高的放大倍率可视化。在体外测定中，单独的成熟卵母细胞从卵巢中分离，并用低渗溶液激活，称为活化缓冲液，这已被证明是概括在体内活化15-17的事件。 这种体外试验使不同的实验条件，包括药理中断，物理处理和基因突变下的钙波易高分辨率可视化。本文展示了成熟的卵子果蝇对体外激活和日准备用于可视化使用GCaMP的钙波ê随后的显微镜。这种方法可以用于测试的 Ca 2+波的起始和控制，并探测下游结果。

<table><tbody><tr><td>Dry active yeast</td><td>Fleischman&amp;rsquo;s</td><td>#2192</td><td></td></tr><tr><td>Dissecting microscope</td><td>Leica</td><td>MZ6</td><td>Various will work</td></tr><tr><td>Lamp</td><td>Mircotec Fibre optic</td><td>MF0-90</td><td>Various will work</td></tr><tr><td>Medical wipes</td><td>Kimberly-Clark Corporation</td><td>KLEW3020&amp;nbsp;</td><td></td></tr><tr><td>Marker pen</td><td>Stabilo</td><td>841/46</td><td>OHPen universal, black - available in most stationary shops. Superfine Width 0.4 mm</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; pad</td><td>&amp;nbsp;Genesee Scientific</td><td>59-114 (789060 Dutscher)</td><td></td></tr><tr><td>Cover slip No. 1.5, 22 x 50 mm</td><td>International- No1-VWR Cat=631-0137-22-50mm</td><td>Menzel-Glaser-22-50mm-MNJ 400-070T&amp;nbsp;</td><td></td></tr><tr><td>Halocarbon oil, series 95</td><td>Halocarbon Products Corp.</td><td>Distributor in Europe:</td><td></td></tr><tr><td>Solvadis (GMBH)</td><td></td><td></td><td></td></tr><tr><td>Oil 10 S</td><td>VWR Chemicals</td><td>24627.188</td><td>Can be used instead of Halocarbon oil, series 95</td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11252-00</td><td>Whilst these tool should be clean, use of an autoclave is not necessary, ethanol would be sufficient.</td></tr><tr><td>Probe</td><td>Fine Science Tools</td><td>10140-01</td><td></td></tr><tr><td>Glass pipette</td><td>Fisherbrand,</td><td>FB50251</td><td>Pasteur Pipettes, glass, unplugged, 150 mm length&amp;nbsp;</td></tr><tr><td>Vial</td><td>Regina Industries</td><td>P1014</td><td>GPPS&amp;nbsp; 80 mm x 25 mm</td></tr></tbody></table>

imaging calcium in drosophila at egg activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a 'Ca2+ wave'. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.
In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.

在这里，我们已经演示了如何进行体外活化准备成熟果蝇卵。鸡蛋表达的Ca 2+动力学在蛋激活一个GECI使成像和胚胎发育的开始（ 图1）。应当注意的是，根据不同的GCaMP使用的，特别是肉豆蔻酰基的存在，结果可能有轻微的质的差异13 14。我 ​​们还证明蛋激活期间用于功能的肌动蛋白作用通过加入肌动蛋白聚合的抑制剂，细胞松弛素D，以活化缓冲液（ 图2）14。 一种在蛋激活细胞骨架的要求是守恒的。在C.线虫，受精后，细胞骨架成分改组，以制备单细胞胚胎用于第一不对称分裂21,22。在海胆，cytoskele中断以下激活TAL重新组织已表明通过防止收缩环形成23来影响细胞周期。肌动蛋白在介导的 Ca 2+波，并在蛋活化果蝇其下游功能中的作用仍有待完全阐明。 在果蝇中，可以使用此体外活化实验达到的钙波和细胞骨架的联合可视化。时间序列的多个信道的可获取和细胞内Ca 2+的动力学可以在卵母细胞（ 图3）中的肌动蛋白组织的变化相匹配。 <img alt="图1" src="/files/ftp_upload/54311/54311fig1.jpg" /> 图1:一个单一的 Ca 2+波在果蝇卵的激活时间序列的体外成熟卵果蝇表达UASt- myrGCaMP5继一活化缓冲液（AH）的ddition。在胞质钙的上升2+起源于后极（B）和传播（BF）与约1.5微米/秒的平均速度。波的启动通常是加活化缓冲液的3分钟内观察。以下激活，成熟卵子的细胞内钙水平返回到预活化水平（G，H）。见补充电影1（总时间33分钟）。比例尺= 50微米。最大投影为40μm。 <a href="https://www.jove.com/files/ftp_upload/54311/54311fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54311/54311fig2.jpg" /> 图2:加入细胞松弛素D的活化缓冲摄动的Ca2+波传播。时间序列的体外成熟卵果蝇表达UASt- myrGCaMP5下加入含10微克/毫升松弛素D终浓度（AH）的激活缓冲区。虽然钙波在后极（A）中发起的，它受到损害，并且不到达卵子（F）的前（白色箭头表示波的前部）。没有进一步的 Ca 2+变化在30分钟内观察到的）。见补充电影2（总时间30分钟）。比例尺= 50微米。最大投影为40μm。 <a href="https://www.jove.com/files/ftp_upload/54311/54311fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54311/54311fig3.jpg" /> 图3:达E肌动蛋白和钙离子的联合可视化在果蝇，时间序列体外激活GG成熟果蝇卵表达UASt- myrGCaMP5（青色）和UASP -F-Tractin.tdTomato（品红）以下除了活化缓冲液（AH）。的钙波从后极发起和恢复如在图1。肌动蛋白似乎随时间变化，白色箭头（A 对 H）。缺乏成熟卵的中心的 Ca 2+信号的检测是在图像捕获期间由于样品的移动。比例尺= 50微米。最大投影为40μm。 <a href="https://www.jove.com/files/ftp_upload/54311/54311fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Imaging Calcium in Drosophila at Egg Activation at JoVE.com

Imaging Calcium in Drosophila at Egg Activation

本文介绍了激活卵子在果蝇中可视化钙离子的适应体外协议。

在成像钙<em&gt;果蝇</em&gt;在蛋激活

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. ...

imaging-calcium-in-drosophila-at-egg-activation

Research

JoVE Journal

Developmental Biology

Imaging Calcium in Drosophila at Egg Activation

7.8K 次观看.  University of Cambridge. 本文介绍了激活卵子在果蝇中可视化钙离子的适应体外协议。 

视频: Imaging Calcium in Drosophila at Egg Activation

Upright Imaging of Drosophila Egg Chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

直立成像<em&gt;果蝇</em&gt;蛋钱伯斯

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

科研

发育生物学

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

在气味诱发反应的钙成像果蝇触角叶

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

神经科学

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

早期果蝇胚胎内免疫组织化学和RNA原位分布的方案

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...

Tracking Calcium in the Early Developmental Phases of C. elegans

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode Caenorhabditis elegans provides several unique advantages for the study of the OET as it is transparent and has an ordered gonad that produces one mature oocyte every ~23 min at 20 &#176;C. We have modified the genetically encoded calcium indicator jGCaMP7s to fluorescently indicate the moment of fertilization within a living organism. We have termed this reporter &#34;CaFE&#34; for Calcium during Fertilization in C. elegans. The CaFE reporter was&#160;engineered into a safe harbor locus in single copy, has no significant impact on physiology or fecundity, and produces a robust signal upon fertilization. Here, a series of protocols is presented for utilizing the CaFE reporter as an in vivo tool for dissecting the OET and embryogenesis. We include methods to synchronize worms, examine the effects of RNAi knockdown, mount worms for imaging, and to visualize calcium in oocytes and embryos. Additionally, we present the generation of additional worm strains to aid in this type of analysis. Demonstrating the utility of the CaFE reporter to visualize the timing of fertilization, we report that double ovulation occurs when ipp-5 is targeted by RNAi and that only the first oocyte undergoes immediate fertilization. Furthermore, the discovery of single-cell calcium transients during early embryogenesis is reported here, demonstrating that the CaFE reporter persists into early development. Importantly, the CaFE reporter in worms is simple enough to use for incorporation into course-based undergraduate research (CURE) laboratory classes. The CaFE reporter, coupled with the ordered gonad and ease of RNAi in worms, facilitates inquiry into the cell-cell dynamics required to regulate internal fertilization and early embryogenesis.

Here, we present protocols and tools for visualizing calcium during fertilization and early embryogenesis using a genetically encoded calcium reporter expressed in the germline of the model nematode C. elegans.

Calcium is an important signaling molecule during the oocyte-to-embryo transition (OET) and early embryogenesis. The hermaphroditic nematode ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

前体钙成像用于可视化大脑对果蝇内分泌信号的反应

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

在Vivo光学钙成像学习诱导突触可塑性在果蝇黑色素增生

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

通过钙成像监测 果蝇 的癫痫样事件

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals worldwide. Despite the clinical importance of epilepsy, the functional analysis of neural activity in the central nervous system is still to be developed. Recent advancements in imaging technology, in combination with stable expression of genetically encoded calcium indicators, such as GCaMP6, have revolutionized the study of epilepsy at both brain-wide and single-cell resolution levels. Drosophila melanogaster has emerged as a tool for investigating the molecular and cellular mechanisms underlying epilepsy due to its sophisticated molecular genetics and behavioral assays. In this study, we present a novel and efficient protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The whole brain is prepared from cac, a well-known epilepsy gene, knockdown flies for calcium imaging with a confocal microscope to identify the neural activity as a follow-up to the bang-sensitive seizure-like behavior assay. The cac knockdown flies showed a higher rate of seizure-like behavior and abnormal calcium activities, including more large spikes and fewer small spikes than wild-type flies. The calcium activities were correlated to seizure-like behavior. This methodology serves as an efficient methodology in screening the pathogenic genes for epilepsy and exploring the potential mechanism of epilepsy at the cellular level.

作者聚焦:对癫痫研究最新进展的技术和发现的见解 

Here, we present a protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The protocol provides a valuable tool for investigating ictal events in adult Drosophila through ex vivo calcium imaging, allowing for exploration of the potential mechanisms of epilepsy at the cellular levels.

离体 癫痫 果蝇 模型的钙成像

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals ...

Calcium Imaging in Individual Neurons Regulating Egg-Laying Behavior in Caenorhabditis elegans

Source: Ravi, B. et al., <a href="https://app.jove.com/v/56911">Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans.</a> J. Vis. Exp. (2018).
This video demonstrates a technique for calcium imaging in individual neurons of behaving Caenorhabditis elegans using genetically encoded fluorescent reporter proteins. It outlines the steps involved in imaging worm movement and measuring reporter fluorescence ratios from neurons to correlate intracellular calcium levels with neuronal activity and behavioral changes during egg-laying.

调节秀丽隐杆线虫产卵行为的单个神经元中的钙成像

1. Strains, Culture Media, and Mounting of Animals

	Grow&#160;C. elegans&#160;worms at 20 &#176;C on standard 60-mm Nematode Growth Medium (NGM) agar ...

在成像钙

摘要

探索更多视频

此视频中的章节

直立成像

在气味诱发反应的钙成像果蝇触角叶

早期果蝇胚胎内免疫组织化学和RNA原位分布的方案

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

前体钙成像用于可视化大脑对果蝇内分泌信号的反应

在Vivo光学钙成像学习诱导突触可塑性在果蝇黑色素增生

离体癫痫果蝇模型的钙成像

离体癫痫果蝇模型的钙成像

离体癫痫果蝇模型的钙成像

调节秀丽隐杆线虫产卵行为的单个神经元中的钙成像

在成像钙

摘要

探索更多视频

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直立成像

在气味诱发反应的钙成像果蝇触角叶

早期果蝇胚胎内免疫组织化学和RNA原位分布的方案

In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

前体钙成像用于可视化大脑对果蝇内分泌信号的反应

在Vivo光学钙成像学习诱导突触可塑性在果蝇黑色素增生

离体 癫痫 果蝇 模型的钙成像

离体 癫痫 果蝇 模型的钙成像

离体 癫痫 果蝇 模型的钙成像

调节秀丽隐杆线虫产卵行为的单个神经元中的钙成像

离体癫痫果蝇模型的钙成像

离体癫痫果蝇模型的钙成像

离体癫痫果蝇模型的钙成像