在开始之前准备： <ul><li>流浪yhird龄果蝇幼虫</li><li> HL3.1（修改血淋巴样）解决方案</li><li> Sylgard（透明硅橡胶）夹层板准备在小（35 × 10毫米）的塑料培养皿使用布伦特和麦凯布（2008年）3描述的方法菜肴。 </li><li>削减夹层引脚短</li><li>刺激电极移液器</li><li>夏普录音移液器</li></ul> HL3解决方案： <ol><li>在解剖和电生理实验，幼虫都沉浸在HL3.1解决方案，其中包含（毫米）：70氯化钠，氯化钾，氯化镁2，10 4碳酸氢钠3，5海藻糖，115蔗糖，5 HEPES，pH值7.2。 </li><li> 氯化钙的浓度添加到HL3.1解决方案提供所需的细胞外Ca 2 +浓度。 <ul><li>幼虫解剖是在低Ca 2 +浓度（以避免肌肉收缩）使用HL3.1 + 0.25毫米氯化钙2，放在冰上。 </li><li>电生理实验通常在HL3.1 + 1毫米氯化钙2，但是这可以从0.4调整- 1毫米的Ca 2 +的要求。 </li></ul></li><li> HL3.1的解决办法是在使用前过滤消毒，并保存在4˚C。 </li></ol>刺激和记录的吸液管的制备： <ol><li>记录和刺激移液器被拉到同一个萨特的P - 2000基于激光的微管使用以下设置拉马：热= 390，长丝= 4 = 35，速度，延迟= 200拉= 0。 </li><li>刺激电极准备薄壁高硼硅玻璃毛细管，略高于切断电机轴突的宽度更大的宽两端。形两端firepolished给顺利完成（使用Narashiga吸管抛光），以尽量减少对神经的损害。刺激电极填充液（HL3.1 + 1毫米的Ca 2 +）。 </li><li>记录电极准备从1.2mm高硼硅玻璃，这是拉，形成了尖锐的吸管（30-60MΩ），充满3M氯化钾。 </li></ol>第1部分：夹层果蝇幼虫<ol><li>三龄幼虫徘徊解剖暴露在体壁的肌肉前面描述4。 </li><li>解剖在HL3.1 + 0.25毫米的Ca 2 +（35 × 10毫米）小硅板。 HL3.1解决方案都必须以麻醉幼虫冰冷解剖和解剖之前和期间，冰。 </li><li>幼虫解剖使用布伦特和麦凯布（2008年），即是修改加入0.25毫米的Ca 2 + HL3.1夹层解决方案，并使用少的短清扫引脚（1〜2毫米长）的电生理实验中所描述的方法显微镜物镜和电极有可能打在一个实验。 </li><li>清扫之后，幼虫的运动轴突被切断。这是通过轻轻地捧着中枢神经系统，所以可以在不破坏肌肉（图1）切断周围神经支配的肌肉后向腹侧神经节略有提高。然后取出大脑。 </li><li>解剖的幼虫准备洗两次与HL3.1 + 1毫米的Ca 2 + 。 </li></ol><img src="/files/ftp_upload/1109/fig1.png" alt="图1" /> 图1显示在切割解剖幼虫的运动轴突所涉及的步骤示意图。中枢神经系统解除钳和运动轴突在大脑底部切割。 第2部分：从幼虫的肌肉细胞的细胞内记录。 <ol><li>广场上的电镜和淹没的准备与HL3.1夹层板+ 1.5毫米的Ca 2 +，所以在接触解决方案是修复浴电极。 </li><li>在第三腹段（A3），所有的实验都进行肌肉6。周围神经支配的肌肉刺激使用吸入电极5。 </li><li>在第一盘的刺激电极的位置，以避免震动，当细胞内的电极到位。刺激电极放置在邻近的运动神经元支配肌肉6，直到切断神经内的玻璃吸管轻轻吸。小心吸力时，不舒展神经。提高吸管轻微，所以它是不碰的肌肉和位置，以便它不切断神经纤维（图2）拉。 <img src="/files/ftp_upload/1109/fig2.png" alt="图2" width="600" height="296" /> 图2：从果蝇幼虫腹段的肌肉和支配运动神经元的示意图。刺激吸管和切断的神经和肌肉6位置的记录电极的位置说明。 </li><li>内与3米氯化钾充满尖锐的微电极细胞的录音。以上的肌肉6第三腹段（A3）的中心位置的记录电极和调整的输入偏移，所以它读取零液。慢慢地降低电极和高光学倍率下的肌肉的方法，直到它触及肌肉表面。观看示波器确认的肌肉已经渗透。静息膜电位应至少-60 mV时，不应使用或动物。现在应该可见零星的微型终板潜力。离开细胞稳定一分钟才开始录制。 </li><li>膜电位的变化是轴突HS - 2A头阶段，Axoclamp 2B放大器和检测记录Clampex v 8.2.0.235（Axon仪器）。 Axoclamp 2B是连接到一台运行pCLAMP软件和工程结合Digidata 1322A接口。 </li><li>细胞记录为3分钟，没有任何刺激来衡量mEJP的反应。痕迹分析，使用Mini分析软件（Synaptosoft，V 6.0.3）和mEJP幅度和频率决定。 </li></ol>神经刺激： <ol><li>到底是一个方形的电压脉冲（0.3毫秒）的强度是足够的，对于这两个运动轴突，一致的反应，以唤起整个肌肉的一系列刺激运动神经切断。 </li><li>刺激产生的主8脉冲发生器，这是编程提供脉冲持续的时间和间隔时间。 </li><li>允许5秒之间在NMJ突触恢复刺激。 </li><li>确定刺激的强度时，开始的最低水平，并多次试验，强度缓慢增加。应在实验中使用的最小刺激强度，结果诱发反应。 </li><li>当达到适当的刺激强度，应该有一个复合EJP和刺激诱发的肌肉收缩。 </li><li>记录至少10个诱发电位的振幅从每一块肌肉和平均。 </li></ol>第3部分：代表结果<img src="/files/ftp_upload/1109/fig3.png" alt="图3" width="600" height="358" /> 图3。代表从6肌肉细胞内记录显示诱发的EJP节段性神经电刺激和零星的微型终板电位，或mEJP的回应。 EJP幅度，在健康的野生型幼虫，如广东小号，6肌肉通常是大约40 mV和1-3之间MV mEJP幅度。

<ol>
	<li>Atwood, H. L., Govind, C. K., 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=Differential+ultrastructure+of+synaptic+terminals+on+ventral+longitudinal+abdominal+muscles+in+Drosophila+larvae.">Differential ultrastructure of synaptic terminals on ventral longitudinal abdominal muscles in Drosophila larvae.</a> J. Neurobiol. 24 (8), 1008-1024 (1993).</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 Neurogenet. 18 (2), 377-402 (2004).</li><li>Estes, P. S., Roos, J., van der Bliek, A., Kelly, R. B., Krishnan, K. S., Ramaswami, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traffic+of+dynamin+within+individual+Drosophila+synaptic+boutons+relative+to+compartment-specific+markers.">Traffic of dynamin within individual Drosophila synaptic boutons relative to compartment-specific markers.</a> J Neurosci. 16, 5443-5456 (1996).</li><li>Brent, J. R., Werner, K. M., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+dissection+of+Drosophila+larvae.">Protocol for dissection of Drosophila larvae.</a> J Vis Exp. , (2008).</li><li>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=Properties+of+the+larval+neuromuscular+junction+in+Drosophila+melanogaster.">Properties of the larval neuromuscular junction in Drosophila melanogaster.</a> J Physiol (Lond). 262, 189-214 (1976).</li><li>Katz, L. C., Shatz, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+activity+and+the+construction+of+cortical+circuits.">Synaptic activity and the construction of cortical circuits.</a> Science. 274, 1133-1138 (1996).</li></ol>

这里所描述的方法提供了一个相对快速和广泛的方式来检测在NMJ突触功能的改变。执行，使用完整的动物体内电生理记录，并执行遗传或药理操作的能力，使果蝇神经递质的生理和遗传方面的调查的一个理想的动物模型。 由于肌肉细胞是非常大的的，有些人可能希望添加一个额外的步骤，为两个电极电压钳（TEVC）记录本议定书。这可以执行相同的幼虫准备在细胞内的电极，?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Small Petri dishes (35 x 10 mm)</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>1008</td>
<td>&nbsp;</td>
<tr>
<td>SYLGARD 182 Silicone Elastomer Kit</td>
<td>&nbsp;</td>
<td>Dow Corning Corporation</td>
<td>3097366-1004</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting microscope</td>
<td>&nbsp;</td>
<td>Carl Zeiss</td>
<td>475002-9902</td>
<td>&nbsp;</td>
<tr>
<td>Light for microscope</td>
<td>&nbsp;</td>
<td>Schott</td>
<td>KLI500</td>
<td>&nbsp;</td>
<tr>
<td>Dissection pins</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-10</td>
<td>&nbsp;</td>
<tr>
<td>pClamp 9 software</td>
<td>&nbsp;</td>
<td>Axon CNS, Molecular Devices</td>
<td>PCLAMP 9 STANDARD</td>
<td>&nbsp;</td>
<tr>
<td>Dissection scissors: 3mm Vannas Spring Scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15000-0</td>
<td>&nbsp;</td>
<tr>
<td>Dumont SS Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11200-33</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11252-20</td>
<td>&nbsp;</td>
<tr>
<td>Thin-walled borosilicate glass capillaries, with filament (1.0 mm, 4 in)</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>TW100F-4</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass capillaries, with filament (1.2 mm, 4 in)</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>1B120F-4</td>
<td>&nbsp;</td>
<tr>
<td>Sutter P-2000 Laser Based Micropipette Puller </td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>Model P-2000</td>
<td>&nbsp;</td>
<tr>
<td>Pipette polisher</td>
<td>&nbsp;</td>
<td>Narishiga</td>
<td>MF-83</td>
<td>&nbsp;</td>
<tr>
<td>Axon HS-2A head stage</td>
<td>&nbsp;</td>
<td>Axon CNS, Molecular Devices</td>
<td>Model HS-2A</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulators</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>MP-85</td>
<td>&nbsp;</td>
<tr>
<td>Axoclamp 2B amplifier</td>
<td>&nbsp;</td>
<td>Axon CNS, Molecular Devices</td>
<td>AXOCLAMP 2B</td>
<td>&nbsp;</td>
<tr>
<td>Clampex Software</td>
<td>&nbsp;</td>
<td>Axon CNS, Molecular Devices</td>
<td>v 8.2.0.235</td>
<td>&nbsp;</td>
<tr>
<td>Mini analysis software. v 6.0.3</td>
<td>&nbsp;</td>
<td>Synaptosoft</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Brownlee Precision Amplifier</td>
<td>&nbsp;</td>
<td>Brownlee</td>
<td>Model 410</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Baker</td>
<td>4058-01</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>p-9333 </td>
<td>&nbsp;</td>
<tr>
<td>NaHCO3</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>s6297-1kg</td>
<td>&nbsp;</td>
<tr>
<td>Trelahose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>TO167</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>bp220-212 </td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>h-3375</td>
<td>&nbsp;</td>
<tr>
<td>MgCl-6H2O</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>m2670-1kg </td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>c79-500</td>
<td>&nbsp;</td>
<tr>
<td>Master-8 Pulse Generator</td>
<td>&nbsp;</td>
<td>A.M.P.I</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vibration table for electrophysiology set up</td>
<td>&nbsp;</td>
<td>Technical manufacturing corporation</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Faraday Cage</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

electrophysiological methods for recording synaptic potentials from the nmj of drosophila larvae

在这个视频中，我们描述记录果蝇幼虫神经肌肉接头处（NMJ）突触传递的电生理方法。幼虫神经肌肉系统是一个模型突触，突触生理学和神经递质的研究，是一个有价值的研究工具，已定义遗传学和实验操作。幼虫可解剖体壁肌肉，中枢神经系统，以及末梢神经暴露。果蝇的肌肉和他们的神经支配模式是很好的特点，很容易和肌肉细胞内记录访问。单独的肌肉可以识别8腹节安排在A2段重复的模式，每30肌肉内的位置和方向 - A7。解剖果蝇幼虫薄和个人肌肉的运动神经元轴突束可以透可视化<sup&gt; 1</sup&gt;。视觉识别或操纵基因在特定的组织产品标签的靶细胞，可用于转基因构造。幼虫，兴奋交界电位（EJPs）产生响应的运动神经元突触释放的谷氨酸水泡。在解剖幼虫，可以记录EJP，在肌肉与细胞内的电极。电机被切断后的腹侧神经节，绘制成玻璃吸管轻轻吸电极刺激神经元动作电位可以人为诱发。这些运动神经元有明显的发射时的刺激阈值，而当他们同时火，它们产生在肌肉的反应。整个NMJ突触传输的信号可以被记录在肌肉的运动神经元支配。 EJPs和微型兴奋交界电位（mEJPs）被视为膜电位的变化。在室温下的电生理反应记录修改最小的血淋巴样的解决方案<sup&gt; 2</sup&gt;（HL3），包含5毫米的镁<sup&gt; 2 +</sup&gt;和1.5 mm钙<sup&gt; 2 +</sup&gt;。诱发EJPs幅度的变化可以表明突触功能和结构的差异。数字化录音分析mEJP EJP幅度，频率和振幅，和量子内容。

Watch this Scientific Journal Video about Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae at JoVE.com

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

在这里，我们描述了测量在果蝇幼虫神经肌肉接头处突触传递的电生理方法。发起人为诱发释放刺激运动神经元轴突，并通过NMJ传输，可在肌肉引起突触后反应测量。

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. ...

electrophysiological-methods-for-recording-synaptic-potentials-from

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

20.9K 次观看.  Columbia University College of Physicians and Surgeons. 在这里，我们描述了测量在果蝇幼虫神经肌肉接头处突触传递的电生理方法。发起人为诱发释放刺激运动神经元轴突，并通过NMJ传输，可在肌肉引起突触后反应测量。

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

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

从3龄果蝇幼虫的成虫光盘夹层

科研

生物学

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

膜片钳从花萼电容录音方法

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

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

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited.

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.

Channelrhodopsin2介导的突触电位刺激果蝇神经肌肉接头

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available ...

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

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

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. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. 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.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

在电生理记录果蝇胚胎

Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is still much to learn from this model preparation. In understanding the history one can appreciate why even today this NMJ still offers a rich playground to address questions regarding pre- and post-synaptic function and plasticity. The viability and ease of access to the terminal for intracellular as well as extracellular electrophysiology and imaging are significant advantages. The mechanisms behind the modulation of vesicular kinetics and fusion within the high- and low-output terminals are begging for investigation. The preparation also offers a testable model system for computational assessments and manipulations to examine key variables in theoretical models of synaptic function, for example calcium dynamics during short-term facilitation. The synaptic complexity of active zone and statistical nature of quantal release is also an open area for future investigation both experimentally and computationally.

The opener muscle of the crayfish leg is presented for its historical importance and experimental versatility in muscle phenotype, synaptic physiology and plasticity.

小龙虾开瓶器肌肉NMJ的历史观和生理学示范

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

在完整的果蝇幼虫体内的亚细胞分辨率成像

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to study the functions and evolution of bacterial symbionts in their host. As most symbionts cannot be cultured in vitro, methods to isolate an adequate quantity of bacteria for genome sequencing are very important. In the whitefly Bemisia tabaci, a number of endosymbionts have been identified and are predicted to be of importance in the development and reproduction of the pests through multiple approaches. However, the mechanism underpinning the associations remains largely unknown. The obstacle partially comes from the fact that the endosymbionts in whitefly, mostly restrained in bacteriocytes, are hard to separate from the host cells. Here we report a step-by-step protocol for the identification, extraction and purification of endosymbionts from the whitefly B. tabaci mainly by dissection and filtration. Endosymbiont samples prepared by this method, although still a mixture of different endosymbiont species, are suitable for subsequent genome sequencing and analysis of the possible roles of endosymbionts in B. tabaci. This method may also be used to isolate endosymbionts from other insects.

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Here, we present a protocol to isolate endosymbionts from the whitefly Bemisia tabaci through dissection and filtration. After amplification, the DNA samples are suitable for subsequent sequencing and study of the mutualism between endosymbionts and whitefly.

从白蛉提取内分泌物的方法<em&gt; Bemisia tabaci</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...