KB是由美国国立卫生研究院授予GM54544支持。

第1部分:设备和用品<ol><li> 果蝇胚胎的电生理记录，首先需要在胚胎夹层技术的能力，这是在另一朱庇特的视频。 </li><li> 果蝇胚胎的电生理记录使用标准的膜片钳记录配置。膜片钳记录设备和软件适用于许多其他准备工作也适合记录果蝇胚胎。由于果蝇胚胎粘过程中剥离的盖玻片，录音室应接受盖玻片。编制和方式，它是安装在盖玻片厚度需要一个优秀的DIC的光学和直立显微镜长工作距离镜头。 </li><li>两种类型的沐浴解决方案用于电生理记录; 1）"标准"（月和1月，1976a）或"修改标准"（Broadie 2000）salines，根据记录在其他无脊椎动物系统常用的解决方案，以及2） "血淋巴样"（HL）的salines（Stewart等，1994），标准生理盐水和在果蝇血淋巴测量离子浓度之间的妥协。应该指出，这些salines都不准确模仿的血淋巴中的离子浓度测量，记录不利（Broadie 2000）。标准生理盐水（毫米）:135氯化钠，氯化钾，氯化镁2，1.8氯化钙，72蔗糖，5工商业污水附加费，pH值7.2。 </li></ol>第2部分:配置记录<ol><li>生理实验，解剖的胚胎准备是放置在一个小有机玻璃录音室（&lt;0.5毫升），在透射光看一个堂堂正正的复合显微镜装有微分干涉对比（Nomarski）光学和长工作距离40 - 100X（通常40X）浸水的目标（Broadie和巴特，1993）。录音通常低于室温（16-22℃），在高温下难度加大，（22 ±℃）。我们实现冷却的房间，而不是冷却preapartion。 </li><li>补丁移液器可拉从各种纤维填充眼镜（如硼硅玻璃，外径1毫米），提示应火抛光到最后的5-10兆欧电阻。一个氯化银地线是摆在洗澡。信号被放大使用一个膜片钳放大器（如Axopatch - 1D，Axon仪器），在2-10赫兹与8极Bessel滤波器过滤，要么上线采样，数字或储存供日后分析。 </li><li>传统的膜片钳记录，最常见于18 °拉硼硅玻璃纤维填充的补丁移液器C（提示热抛光〜1微米的ID）。目前的纪录是由确定多核肌纤维，在电压钳（标准控股潜力-60 -80毫伏）（Broadie和巴特，1993 - D; Broadie等，1997;。Fergestad等人，1999年;费瑟斯通等，2000）。补丁移液器解决方案包含（毫米）:120氯化钾，氢氧化钾，20 4 0.25氯化钙氯化镁2，2，5 EGTA，4钠三磷酸腺苷，36蔗糖和5工商业污水附加费，在pH 7.2缓冲。记录电极回填。 </li></ol>第3部分:肌肉记录<ol><li>原则上，任何胚胎的肌肉可能会被记录在此准备。在实践中，大多数录制集中在大，腹纵肌在最内层的肌肉层（巴特，1990年; Broadie和巴特，1993 - C）。记录也被从这个肌肉层的背侧和外侧的肌肉（Kidokoro西川，1994年;奥尔德等人，1995年;西川和Kidokoro，1995年，鲍姆加特纳，1996）。然而，大多数实验的重点放在四纵，腹肌肉组（6,7,12,13），尤其是前腹部肌肉（A2 - A3）6。 </li><li>无酶处理之前需要肌肉幼胚（&lt;16小时的机场快线）的录音。然而，肌鞘在以后的胚胎发展（&gt; 16小时机场快线），必须用胶原酶（IV型胶原酶，1毫克/毫升的二价阳离子免费生理盐水，在室温下0.5-2分钟）之前，修补记录中删除。酶处理后，对肌肉的密封电阻通常&gt; 1GΩ。 </li><li>膜片钳记录，可从使用的标准技术胚胎肌肉（Broadie和贝特1993 - C; Broadie等1994年，1995年，1997年;西川和Kidokoro，1995年）。任何标准的变化 - 膜片钳细胞贴附，由内向外，外出来 - 可以记录在肌肉或孤立的肌肉膜补丁单通道活动。单谷氨酸受体通道活动（-60毫伏〜12 PA）一般是观察到​​的自发兴奋交界处，在全细胞模式记录电流（sEJCs）下降阶段。 </li><li>大部分记录是在全细胞记录配置掩膜，实现了从细胞连接状态，轻松地与轻微吸或电气的"嗡嗡"。无论是在电压钳（典型值-60至-80 mV的潜力）或电流钳配置的肌肉可以记录。在年轻的胚胎（&lt;13小时机场快线）的肌肉可以不被有效地电压，钳位由于在（Broadie和巴特，1993年）胚胎肌管之间广泛的耦合，这是通常不会在以后的一个问题（14 +小时机场快线）的发展阶段（上田Kidokoro，1996年）。 </li><li>制霉菌素穿孔膜片钳记录可用于验证标准的全细胞技术（Broadie和巴特，1993）获得的记录。穿孔的解决方法是如下:10毫克聚醚F - 127（P - 1572的分子探针）是在20毫升DMSO溶解，10毫克制霉菌素溶解在这种解决方案使股票。 100μL的股票将被添加到5毫升的过滤移液器解决方案，使录音解决方案。 &lt;3分钟后，密封形成全细胞电流通常获得。串联电阻明显增加，在大多数情况下，细胞可以发展的漏电电流（Broadie和巴特，1993）。 </li><li>全细胞电压门控目前在成熟的胚胎和幼虫的肌肉组成的五个突出的组件（吴和格兰，1985年; Zagotta等1988; Broadie和巴特，1993b。）:一个内向钙电流（ 我钙）和4个外向钾电流（Tsunoda和Salkoff，1995年），包括两个快速失活电流，电压门控（一）和钙依赖性（CF），和两个延迟，非失活电流，电压门控（我K）和钙依赖性（CS）。可以用离子替代实验，解剖这些电流在电压钳全细胞的反应（Broadie和巴特，1993年b）。 </li></ol>第4部分:神经肌肉接头的刺激和记录<ol><li> NMJ刺激最常见的和可靠的方法是使用玻璃吸电极对周围神经节段性。吸电极可以拉出各种电极的玻璃纤维填充（1-1.5微米外径）和应火抛光，以达到所需的配置（内径〜5微米）。吸管轻轻吸绘制成一个适当的节段性运动神经的一小部分（&lt;50微米），形成一个密封。 </li><li>这是常见的中枢神经系统的退出点附近的神经回路，以刺激完整的神经。这种方法允许容易刺激完整的准备，但有一个缺点就是自发的中枢神经系统引起的活动可以发生在应用刺激范式叠加。另一种方法是刺激切断运动神经。要做到这一点，如果中枢神经系统被删除，但此过程往往舒展神经，可能会损坏。用锋利的玻璃微电极的神经也可能被削减，但这个过程是乏味和困难。这是建议，记录完整的准备。 </li><li>刺激可应用于各种范式。简要刺激（0.2-1毫秒）最好的作品和刺激的强度（一般为1-5 V）将取决于吸配置，必须为每个单元实验确定。最好是通过设置的刺激强度略高于（+1-2伏）阈值阈上刺激的运动神经。如上所述，诱发NMJ响应的记录片夹住肌肉。冲击神器，就可以大大减少使用一个独立的虚拟地面。 </li><li>一个不可靠的替代标准吸电极神经刺激直接刺激中枢神经系统（西川和Kidokoro，1995年）。腹侧神经节〜2微安的强度和1998年1〜2毫秒的持续时间交付（Dietcher等）的正脉冲的中间插入一个微电极充满3-4米氯化钾（或KAC）。电极尖端应在最近的神经纤维的刺激所需hemisegment。突触传递的是记录片夹紧在标准配置中的肌肉中。 </li><li>全细胞膜片钳记录整个分化的胚胎NMJ访问。兴奋交界电流（EJC的）录音，信号记录与一个标准的膜片钳放大器，过滤（通常在1-2千赫），使用模拟到数字接口的转换为数字信号，并用计算机获得的使用相应的软件。 </li></ol>第5部分:神经肌肉接头处的化学和药物的应用<ol><li>任何小，带电分子可以有效iontophoresed到胚胎NMJ（Aravamudan等，1999;。Fergestad等1999年，2001年）。拉Iontophoretic移液器可为特定的配置，变量期间的应用，使用电流脉冲的正面或​​负面。至关重要的是要防止漏之间的PU目前有一个适当的反对支持lses（Broadie等1993d;费瑟斯通等人，2002年，2005年。Rohrbough等2007）。这种支持电流的大小会随吸管配置，应试验确定。 </li><li>许多实验已经完成与NMJ神经递质，L -谷氨酸（月和1月，1976b），使用一个100mm原液（味精盐，pH值8-9）（Broadie和巴特，1993c）。谷氨酸受体（gluR）映射（Broadie和巴特，1993d）已用于高电阻（100〜200MΩ）iontophoretic移液器，低电阻移液器（20-50MΩ）用来估算总gluR响应（Broadie等人。 1995年，1997年;费瑟斯通等2002年，2005年; Rohrbough等2007）。负电流（〜10 NA）的工作，以及与一个小的，积极的支持，目前，防止泄漏，短脉冲（0.1-1 MS）。 </li><li>不带电电荷的分子可以通过施加压力弹射（Fergestad和Broadie，2001年;费瑟斯通等人，2001年）。压力弹射吸管（内径&lt;5微米），包含解决方案的应用，定位接近（&lt;10微米）从神经末梢。弹出的解决方案可应用于使用picospritzer（5-10 PSI）或其他加压空气源（费瑟斯通等，2002）。在长时间的实验，录音室（体积&lt;0.5毫升），应连续灌注正常浴生理盐水（0.1 - 0.2毫升/秒）。 </li><li>许多实验已经完成与高渗生理盐水触发突触小泡的融合（Broadie等，1995; Aravamudan，等，1999;。Fergestad等，1999;。费瑟斯通，等人，2000年，2002年）。与毒素，如谷氨酸受体和黑寡妇蜘蛛毒液含有latroinsectotoxin（Broadie等，1995）636 argiotoxin对其他实验已经完成。 </li></ol>第6部分:互补性神经肌肉交界处重要的成像技术<ol><li>可能是突触囊泡动力学监测与亲脂性荧光染料（Fergestad和Broadie，2001年; Rohrbough等人，2004年）stryl。囊泡的分布，可以直接观察，内吞作用和胞吐率测量。几个变种已与不同的荧光性质（FM1 -​​ 43，荧光; FM4 - 64，罗丹明）。修改原染料产生了改变的疏水性能的产品，并因此具有不同的冲刷动力学;例如，无瑕- 10不赞成从膜快得多。 </li><li>标签与stryl染料胚胎NMJ，10微米FM1 -​​ 43可装载使用外钾离子升高（50-90毫米）"（Fergestad和Broadie，2001年）时间为1-5分钟。一个更符合生理的方法，可实现通过刺激神经吸电极。的筹备工作，然后反复冲洗，在 Ca 2 +无缓冲5分钟，并保留荧光（代表染成突触囊泡）测量。要测量胞吐，可以卸载的染料，无论是使用高架钾或神经电刺激。 </li><li>已经产生的融合蛋白与GFP的变种，允许胚胎NMJ突触多色标签（品牌，1999年，张等人，2002年）。许多转基因表达GFP标记的蛋白质，包括一般膜标志物（如mCD8 - GFP），突触后标志物（如沙克尔- GFP GluRIIA - GFP），细胞骨架（如肌动蛋白GFP，moesin - GFP，微管蛋白GFP）和线粒体标记（如细胞色素C - GFP）和突触小泡标记（如synaptobrevin - GFP，突触- GFP;张等，2002）。 </li><li> GFP记者标签车厢，是高度动态的，可用于评估发展特点或突变表型（费瑟斯通和Broadie，2000年）。漂白，然后观察荧光恢复（FRAP）已被证明是可行的，至少在幼虫果蝇NMJ（张等人，2002年;燕和Broadie，2007年）， 。一个忠告，然而，这些标记中的一些不全力营救空突变体，甚至可能会改变正常NMJ生理。 </li></ol>第7部分:从中央的运动神经元的记录<ol><li> SOMA位置和投影形态（巴特和马丁内斯阿里亚斯，1993年，古德曼和美国能源部，1993年;兰德格拉夫等1997）的基础上，许多胚胎神经元可单独确定。此外，GAL4/UAS系统（品牌和Perrimon，1993年）目标GFP的表达，以可视化识别的神经元的人群（伯恩斯等，1998）。迄今记录的研究都集中在一组5个肤浅的，在胚胎腹侧神经线（VNC）的背神经元;四个（ACC和RP1 - 4）的运动神经元和中间神经PCC（贝恩斯等，1998年，1999年，2001年。 Rohrbough和Broadie，2002年;费瑟斯通等1995）。 </li><li>确定神经元的胞体全细胞膜片钳记录电极。 VNC neurolemma鞘必须首先简短的蛋白酶（贝恩斯和巴特，1998年的重点治疗中删除;贝恩斯Ë吨，1999年;。Rohrbough Broadie，2002年）。背侧神经细胞组织接触使用了大直径（〜20微米）修补吸管含有0.5-1％的蛋白酶（类型14（Sigma公司），在录音生理盐水）。为1-2分钟，直到鞘破裂（费瑟斯通等，1995）中枢神经系统的护套材料绘制的枪头吸。为了确认身份在录音，路西法黄色（二钾盐，1-2毫克/毫升）的细胞，可以被添加到该修补程序解决方案，以可视化中的地位和细胞形态。 </li><li>标准电压和电流钳全细胞记录可以（Rohrbough和Broadie，2002年;。费瑟斯通等，2005）。外部录音生理盐水（毫米）:140氯化钠，氯化钾，2 4 氯化钙 ，氯化镁2，5 HEPES，10蔗糖，2氢氧化钠（pH值7.2）。该修补程序解决方案包含（毫米）:140 K -醋酸钠分子的ATP，2 MgCl 2的 0.1氯化钙，10 HEPES，1.1 EGTA，2〜6 KOH（pH值7.2）。控股潜力-60毫伏电压钳记录是在18 ° C。在-45至-65 mV的范围内典型的未经调整的静息电位（-52.9 ± 7.5 mV的，平均± SD，N ​​= 78）。 </li><li>现有的实验揭示电压门控的离子电流（贝恩斯和巴特，1998年），重复动作电位，突触介导的活性和门控受体激动剂的反应（伯恩斯等，2001）。运动神经元突触输入既快速AP介导快速兴奋性突触电流（ES细胞），并发生数次或每分钟（贝恩斯等兴奋性输入的定期，持续（1秒）发作的形式记录，1999;伯恩斯等，2001）。 </li><li>细胞SOMA回应iontophoretically应用乙酰胆碱（ACH）和抑制发射机GABA（贝恩斯和巴特，1998年;。费瑟斯通等人，2005年）。可以iontophoretically应用乙酰胆碱的神经元胞体，通过尖锐的包含卫生署 2 0 100MM乙酰胆碱的微电极，在pH值4-5赞成激动剂离子导入正电流。 </li></ol>代表性的成果腹侧纵肌6段A2 - A3是最普遍使用的用于记录的目标（Broadie和巴特，1993 - D; Harrison等人1994年，1995年斯威尼在人; Renden等2001;黄等人，2006年; Mohrmann等2008）。在成熟的胚胎和刚孵出的第一龄幼虫（20-21小时机场快线在25 ° C =孵化）6，肌肉全细胞电容通常是25-30 pF的输入阻抗20-40MΩ，与发育年龄而改变。作为一个功能的年龄最大的NMJ突触电流范围从几十picoamps（13-14小时机场快线）1，仅为2nA（20-21小时机场快线），迅速增加。谷氨酸门控电流直接的措施，产量最大的picoamps数百（13-14小时机场快线）1.5 4nA刚孵化前的反应。通常&lt;5 mV的系列电阻误差（总电流X系列电阻），但在成熟，预孵化的胚胎，可高达10 + MV，因此建议串联电阻补偿。平均直径10-30微米和40-80微米的平均长度的胚胎肌肉，似乎显示接近理想的空间钳参数。细胞阻断时间常数（R 系列 XC 细胞 ）平均不到0.25毫秒。 在成熟的胚胎和刚孵出的幼虫，背运动神经元的人口已受到较为有限录音。神经元胞体记录显示在1.8-2.5 PF（2.0 pF的平均），40-60系列电阻MΩ〜5GΩ（贝恩斯和巴特，1998年，斯通等人，2005年）和输入电阻范围内的全细胞电容。显然，这些细胞生长在规模和复杂性在胚胎发育成熟，整个幼虫发育继续增长。在第三龄，全细胞电容为17-20 pF的串联电阻是25-37MΩ输入电阻平均〜900MΩ（Rohrbough Broadie，2002年，理查德伯恩斯，个人通讯）。离子电导增加整个开发过程中，但仍然相当恒定的电流密度（PA / PF）。在成熟胚中，既快速ESC事件和长期平均〜500毫秒（贝恩斯和巴特，1998年，斯通等人，2005年）的电流，平均幅度75 - 100pA的突触电流。在胚胎中，这些持续的活动，平均每分钟3-5次，每分钟增加至〜20首龄（理查德伯恩斯，个人通讯）。需要注意的是果蝇的神经元形态的复杂性和膜性能使得几乎不可能正确钳位电压超出了SOMA细胞。因此，人们应该谨慎解释的录音。

The authors have nothing to disclose.

果蝇胚胎的电生理记录需要手工操作和解剖。该制剂的健康，以及随之而来的录音质量，取决于一个能够迅速而整齐地准备录制脆弱的胚胎组织，然后执行实验。实验者应精通胚胎解剖和膜片钳电，然后再尝试一次都解决。 录音可以做的"修订标准"或"血淋巴样"（HL），salines几个变种之一。这两个生理盐水类之间的主要区别是:1）Na +浓度（与70毫米HL 120-135毫米?...

electrophysiological recording in the drosophila embryo

果蝇胚胎发育和功能的神经科学的研究是首屈一指的遗传模型。传统上，这些领域都相当彼此孤立的，很大程度上是独立的历史和科学界。然而，这些通常是不同的领域之间的接口是发展计划的基本功能的电子信号的属性和功能的化学突触在神经回路形成的最后阶段分化的收购。该接口是一个极为重要的领域进行调查。在果蝇中，这些功能开发阶段发生在胚胎发育的最后三分之一的&lt;8小时期间（在25 ° C） 。后期发展时期，长期被视为棘手的调查，由于一个艰难的，不透水的表皮角质层的沉积。一个突破性的进展是，可以在本地应用的角质层，使后期胚胎的控制清扫手术，聚合水胶中的应用。胚胎背侧纵切口，可以平放，露出腹侧神经线和体壁肌肉的实验研究。全细胞膜片钳技术，然后可以记录单独识别的神经元和躯体肌肉。这些记录配置已被用来跟踪的外观和成熟的离子流和行动中的神经元和肌肉的潜在传播。影响这些电气特性的遗传突变体进行了表征，揭示了离子通道及相关的信号复合物的分子组成，并开始探索功能分化的分子机制。一个特别的重点一直是突触连接的大会，无论是在中枢神经系统和外围。谷氨酸能神经肌肉接头（NMJ）是最容易结合光学成像和电生理记录。玻璃吸电极是用于刺激周围神经兴奋结的电流电压钳位肌肉（EJC）录制。此记录已配置使用，以图表的突触功能的分化，并跟踪突触前谷氨酸释放属性的外观和成熟。此外，突触后性能可以独立检测，直接通过谷氨酸iontophoretic或施加压力的肌肉表面，测量谷氨酸受体字段的外观和成熟。因此，可以监视元素前和突触后单独或合并在胚胎突触。该系统已大量用于分离和鉴定基因突变体，损害胚胎的突触的形成，从而揭示了突触连接和功能的突触信号特性的规范和分化的分子机制。

Watch this Scientific Journal Video about Electrophysiological Recording in the Drosophila Embryo at JoVE.com

Electrophysiological Recording in the Drosophila Embryo

从电生理记录<em&gt;果蝇</em&gt;胚胎允许发展肌肉和神经元的电性能，以及在谷氨酸能神经肌肉接头及中枢胆碱能和GABA能突触功能突触的特性分析。

在电生理记录果蝇胚胎

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

electrophysiological-recording-in-the-drosophila-embryo

Research

JoVE Journal

Biology

Electrophysiological Recording in the Drosophila Embryo

11.4K 次观看.  University of Illinois. 从电生理记录果蝇胚胎允许发展肌肉和神经元的电性能，以及在谷氨酸能神经肌肉接头及中枢胆碱能和GABA能突触功能突触的特性分析。

视频: Electrophysiological Recording in the Drosophila Embryo

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

在鸡胚神经诱导的含量为

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

科研

生物学

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

电生理方法记录从果蝇幼虫NMJ突触潜力

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

收获和准备果蝇胚胎的电生理记录和其他程序

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

研究有丝分裂的显微注射技术果蝇合胞体胚胎

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

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 ...

Ambulatory ECG Recording in Mice

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although the surface ECG is utilized for short-term measurements of waveform intervals, it is not practical for long-term studies of heart rate variability or the capture of rare episodes of arrhythmias. Implantable ECG telemeters offer the advantages of simple surgical implantation, long-term recording of electrograms in ambulatory mice, and scalability with simultaneous recordings of multiple animals. Here, we present a step-by-step guide to the implantation of telemeters for ambulatory ECG recording in mice. Careful adherence to aseptic technique is required for favorable survival results with the possibility of implantation and recording from weeks to months. Thus, implantable ECG telemetry is a valuable tool for detection of critical information on cardiac electrophysiology in ambulatory animal models such as the mouse. 

Telemetric ECG has emerged as an essential tool in evaluating animal models for cardiac arrhythmias and sudden cardiac death. Here, we present a stepwise guide to telemetric ECG recordings for application in long-term ambulatory ECG monitoring in mice.

在小鼠体内的动态心电图记录

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although ...

Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28&deg;C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28&deg;C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37&deg;C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28&deg;C for further incubation. We find that the initial incubation at 28&deg;C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e.g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.

Reliable method for highly efficient in vitro expression and subsequent electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T).

优化转染的HEK - 293T细胞中的表达和重组的电压门控离子通道的电生理记录策略

The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

在早期鸡胚跟踪形态组织变形

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

鼠胚体外发育无血清全胚胎培养系统

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...