由 NIH 资助 R01 MH 099557

1. 制作记录电极 <ol> <li> 拉动玻璃电极 <ol> <li> 使用以下协议为微电极拉出器 (参见 材料表 ): <ol> <li> 1 行: 热510拉速30时间 250;2行: 热490拉速30时间 250. 
			注: 时间单位对应于每单位0.5 毫秒;其他单位是相对的。在进行坡道试验后, 应调整每根灯丝的热量值. </li>
		</ol> </li> <li> 使用显微镜 (35x 放大倍数) 来确保所拉电极的内径在 7-10 和 #181 的范围内; m ( 图 1A )。将毛细血管储存在密闭的容器内, 防止尘埃积聚. </li>
	</ol> </li> <li> 火抛光 <ol> <li> 火波兰毛细管 (80-90% 的最大热值为 1-2 s) 使用微锻 (见 材料表 )。确保抛光电极的最终内径为5和 #181; m ( 图 1A ) 27 . </li>
	</ol> </li> <li> 弯曲 注: 两个弯曲是为了将电极放置在高放大目标下的肌肉顶部。
	<ol> <li> 使用 图 1B 中显示的设备。将电极固定在机械手上, 并将刀尖放在灯丝上, 而不是触摸它. </li>
		<li> 将热值设置为最大值的 60-70%, 然后按下微锻的踏板进行 1-2 秒 (请参阅 材料表 ) 来加热灯丝。使用 L 形针轻轻地拉下电极的尖端 ( 图 1B 放大)。使折弯在大约 90 o ( 图 1C ) 上. </li>
		<li> 用镊子将电极用手握住火炬的火焰, 并使第二个弯曲距离第一个弯曲处 7-10 mm, 并以大约 120 o ( 图 1C ) 的角度进行. </li>
	</ol> </li> </ol> <img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56493/56493fig1.jpg"/> 图1。微制作的最后步骤. ( A ) 在拉和火抛光后的电极提示。( B. 1 ) 用于尖端弯曲的设置。( B. 2 ) 在右边显示了已装箱区域的放大。电极和灯丝是固定的, 这样电极尖端就会稍稍在导线上方, 而不接触。( C. 1 ) 记录电极。( C. 2 ) 在右边显示了已装箱区域的放大。第一个折弯有大约90和 #176 的角度; 和第一个弯曲和电极尖端之间的距离大约是1毫米. 刻度条 = 3 mm. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56493/56493fig1large.jpg" target="_blank">请单击此处查看此图的较大版本. </a> 
 2. 其他准备步骤 <ol> <li> 准备 heamolymph 样 (HL3) 解决方案 (mM):70 氯化钠, 5 氯化钾, 20 氯化镁 2 , 10 NaHCO 3 , 5 海藻糖, 115 蔗糖, 5 HEPES, 1 mM CaCl 2 ; 调整 pH 值为 7.3-7.4. 把溶液放在冰箱里, 每周都要新鲜. </li>
	<li> 使刺激吸管与记录玻璃吸管的方式相同, 但不需要对其进行弯曲. 
	注: 在 28 和 27 中详细介绍了刺激电极的制备方法。火焰抛光后的最终直径应在 5-7 和 #181 的范围内; m. </li>
	<li> 在连接到注射器的微电极支架上插入刺激吸管. </li>
	<li> 从小的培养皿 (35 x 10 mm) 上涂覆硅橡胶环氧树脂 (请参阅 材料表 ), 如 28 中所述。
	注: 硅橡胶应在使用前完全硬化. </li>
	<li> 选取一个游荡的三龄幼虫并将其分解为 27 、 28 、 29 、 30 。
	注: 要可视化 boutons, 使用飞应变 CD8-GFP (参见 材料表 ) <ol> <li> 使用镊子 (参见 材料表 ), 从一个小瓶中选取 3 rd 的幼虫. 
		<li> 引脚幼虫, 放置第一针后部和另一针前 (接近口钩). </li>
		<li> 添加 HL3 解决方案 </li>
		<li> 使用弹簧剪刀进行剪切 (请参见 材料表 ) 从顶部针脚到幼虫背侧的底部. </li>
		<li> 引脚幼虫圆角, 在幼虫的左右两侧放置2额外的针. </li>
		<li> 使用镊子清除内脏和气管. </li>
		<li> 用弹簧剪刀切断腹神经线外的神经. </li>
	</ol> </li> </ol> 3。EJCs 的电子录音 <img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56493/56493fig2.jpg"/> 图2。录制设置. 样品 NMJ 固定在硅涂层培养皿 (箭头) 位于直立式复合显微镜的活动阶段, 具有荧光能力, 高放大物镜, 和两个动。显微镜被放在一个防振台上。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56493/56493fig2large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a> 
<ol> <li> 录制 <ol> <li> 在显微镜阶段 ( 图 2 ) 中放置培养皿和准备。在浴缸中插入参考电极. </li>
		<li> 用 HL3 溶液填充记录电极。在10x 目标之下 (参见材料目录 ), 浸洗电极入浴并且安置它在腹部片断 2, 3 或4的肌肉6和7使用微 (参见 材料目录 ) ( 图 3 1 和 2 ). </li>
		<li> 将目标切换到 60x (请参阅 材料表 )。使用 epi-荧光或 DIC 光学来关注感兴趣的领域。将电极尖端放在突触溥 ( 图 3 B. 1-3 ) 上. </li>
		<li> 将电极轻轻地压在肌肉上。过度的压力可能会损害 NMJ 或诱发自发突触活动的增加。确保电极的尖端没有堵塞-如果是的话, 更换它. </li>
		<li> 在放大器、a/d 板和计算机上切换. </li>
		<li> 选择放大器上的电压钳模式 </li>
		<li> 启动购置软件并选择 #39; 无间隙和 #39; 模式. </li>
		<li> 在计算机屏幕上观察 mEJCs 的外观. </li>
		<li> 确保 mEJCs 的振幅在 0.2-0.7 nA 的范围内. 
		注: 较小的 EJCs 表示记录电极有缺陷或未正确定位. </li>
	</ol> </li> </ol> <img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56493/56493fig3.jpg"/> 图3。突触 boutons 的可视化. ( a ) 在10x 缩放 ( 1 ) 下的半段半明图像, 以及显示肌肉6和 7 ( a. 2 , 箭头标记记录电极) 的扩大的盒装区域。缩放条形图 = 50 和 #181; m. ( B ) 突触 boutons 在 果蝇 线上可视化, 使用免疫荧光成像 ( b. 1 ) 或 DIC 光学 ( b. 2 ) 进行基因编码的神经元标记 (CD8-GFP)。图像是采用60x 目标和过滤器立方体的 GFP 成像 (请参阅 材料表 )。Synaptiboutons 用箭头标记, 在 B. 3 中显示了荧光和 DIC 图像的叠加。标尺 = 10 和 #181; m. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56493/56493fig3large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a> 
<ol start="2"> <li> 刺激 <ol> <li> 使用微, 在视觉控制下, 将刺激电极放在轴突支配腹段 2-4. </li>
		<li> 通过拉动连接到电极托架的注射器活塞来施加负压, 以便轴突被拉入电极 ( 图 2 ). </li>
		<li> 打开刺激器。打开隔离装置上的旋钮 (请参见 材料表 ) 以设置零电流, 然后轻轻地将其增大, 直到 EJCs 出现 (或达到阈值为止). </li>
		<li> 在阈的状态下进行刺激, 与 EJCs 观察的阈值相比, 刺激电流增加了大约两倍. 
		注意: 在我们的经验中, 这种刺激强度是最佳的, 以避免动作电位的失败和动作电位的激发。例如, 如果 EJCs 出现在0.2 毫安的刺激电流中, 则在整个实验中使用 0.4 ma 的电流. </li>
	</ol> </li> <li> 密封电阻 <ol> <li> 通过将放大器的电极电阻开关转换为和 #34 来测量记录 macropatch 电极的密封电阻; 密封测试和 #34; 位置。观察 G 和 #937 中的密封电阻值将显示在 #34; 当前和 #34; 窗口. </li>
		<li> 确保密封电阻在 0.5-2 米和 #8486 范围内;。此范围之外的值表示电极未正确抛光或未正确定位. </li>
		<li> 监视整个记录中的密封电阻, 确保在整个实验过程中保持不变. </li>
	</ol> </li> </ol> 4。分析 <ol> <li> 分析使用自定义软件进行解析的记录 (请参见 材料列表 )。
	注: 我们的实验室使用 in-house 软件 Quantan 31 , 它是为检测 EJCs 和 mEJCs 记录的局部 ( 图 4 ) 而定制的。该软件包括一个高斯数字滤波器, 允许在重叠的 multi-quantal 事件中检测量子峰值 ( 图 4A )。其他方法在 17 中进行了描述. </li>
</ol> <img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56493/56493fig4.jpg"/> 图4。量子分析。 通过 Quantan 软件检测 mEJCs ( A ) 和 EJCs ( B )。事件区域以绿色标记, 峰值由红色箭头标记。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56493/56493fig4large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FM+1-43+labeling+of+synaptic+vesicle+pools+at+the+Drosophila+neuromuscular+junction.">FM 1-43 labeling of synaptic vesicle pools at the Drosophila neuromuscular junction.</a> Methods Mol Biol. 440, 349-369 (2008).</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=Drosophila+larval+NMJ+dissection.">Drosophila larval NMJ dissection.</a> J Vis Exp. (24), (2009).</li><li>Imlach, W., 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=Electrophysiological+methods+for+recording+synaptic+potentials+from+the+NMJ+of+Drosophila+larvae.">Electrophysiological methods for recording synaptic potentials from the NMJ of Drosophila larvae.</a> J Vis Exp. (24), (2009).</li><li>Bykhovskaia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+quantal+analysis+more+convenient,+fast,+and+accurate:+user-friendly+software+QUANTAN.">Making quantal analysis more convenient, fast, and accurate: user-friendly software QUANTAN.</a> J Neurosci Methods. 168 (2), 500-513 (2008).</li><li>Huntwork, S., Littleton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complexin+fusion+clamp+regulates+spontaneous+neurotransmitter+release+and+synaptic+growth.">A complexin fusion clamp regulates spontaneous neurotransmitter release and synaptic growth.</a> Nat Neurosci. 10 (10), 1235-1237 (2007).</li><li>Zhong, Y., 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=Altered+synaptic+plasticity+in+Drosophila+memory+mutants+with+a+defective+cyclic+AMP+cascade.">Altered synaptic plasticity in Drosophila memory mutants with a defective cyclic AMP cascade.</a> Science. 251 (4990), 198-201 (1991).</li><li>Delgado, R., Maureira, C., Oliva, C., Kidokoro, Y., Labarca, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+of+vesicle+pools,+rates+of+mobilization,+and+recycling+at+neuromuscular+synapses+of+a+Drosophila+mutant,+shibire.">Size of vesicle pools, rates of mobilization, and recycling at neuromuscular synapses of a Drosophila mutant, shibire.</a> Neuron. 28 (3), 941-953 (2000).</li><li>Melom, J. E., Akbergenova, Y., Gavornik, J. P., Littleton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+and+evoked+release+are+independently+regulated+at+individual+active+zones.">Spontaneous and evoked release are independently regulated at individual active zones.</a> J Neurosci. 33 (44), 17253-17263 (2013).</li></ol>

作者没有什么可透露的。

果蝇代表了研究突触传递的一个有利的模型有机体。在幼体 NMJ 使用了几种记录配置, 包括突触电位的胞内记录、两个电极电压钳33、34和焦点 macropatch 的突触电流记录。在这里描述的突触电流的录音。后一种技术允许精确定量的突触传输在可视化的 boutons。
所描述的协议的成功与否, 关键取决于是否有能力清楚地直观地显示感兴趣的区域, 并定制记录电极的准备工作。因此, 高质量的 DIC 光学, 高倍率的水浸泡物镜, 具有很长的工作距离, 和定制的设备, 用于火焰抛光和弯曲的记录电极是至关重要的。
这种方法的优点是, 它允许监视在记录电极下定位的几个突触的活动。应该注意的是, 然而, 位于电极边缘附近的 boutons 也可能有助于记录活动。因此, 电极的位置以及密封电阻在实验过程中不会发生变化, 这一点至关重要。
监测单个溥的活动的能力可能与最近的成像技术相结合。例如, 在单个活动区域35上的活动的光学检测可以与 EJCs 和 mEJC 的焦点录音相结合, 这可以与电子记录的时间分辨率的光学探测的空间分辨率进行耦合。

果蝇是研究控制突触传递的分子机制的一个很好的模型系统。在果蝇的神经肌肉系统是谷氨酸, 因此,果蝇神经肌肉结 (NMJ) 可以用来研究谷氨酸释放的保守特征。自1月和1月的研究1, 第三龄幼虫已广泛用于研究诱发和自发突触传播通过监测兴奋性结电位 (EJPs) 或电流 (EJCs)。EJPs 通常记录细胞与一个尖锐的玻璃微电极, 它们反映了整个 NMJ 的活动, 包括所有的 boutons 在给定的肌肉纤维的突触。
相比之下, 通过在神经元终端或突触静脉曲张附近定位一个微尖端, 可以记录局部的少量释放点的活动。这项技术最初受聘于 Katz 和 Miledi2, 并成功地使用了一些 NMJ 准备, 包括青蛙3,4,5, 鼠标6,7,8, 甲壳动物9,10,11,12,13,14,15,16, 和果蝇17,18,19,20,21,22,23。这一方法进一步开发了 Dudel, 谁优化 macropatch 重新编码电极24,25。在 Dudel 的实现中, 此技术与松散膜片钳方法26紧密匹配。
果蝇幼虫 NMJ 已明确定义突触 boutons, 和基因编码的神经荧光标记的转基因品系 (见材料表) 是现成的。这些优势使我们能够记录 EJCs 和 mEJCs 从一个选定的突触溥20,21,22。在这里, 我们详细描述这一技术。

<table width="1251">
	<tbody>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Sutter P-97</td>
			<td style="width:288px;">Sutter instrument</td>
			<td style="width:180px;">P-97</td>
			<td style="width:495px;">Microelectrode puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Narishige MF-830</td>
			<td style="width:288px;">Narishige</td>
			<td style="width:180px;">MF-830</td>
			<td>Microforge</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">WPI MF200</td>
			<td style="width:288px;">WPI</td>
			<td>MF200</td>
			<td>Microforge</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Glass capilaries</td>
			<td style="width:288px;">WPI</td>
			<td>B150-86-10</td>
			<td style="width:495px;">Glass capilaries</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microtorch 1WG61</td>
			<td style="width:288px;">Grainer</td>
			<td style="width:180px;">1WG61</td>
			<td>Microtorch</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:288px;">Dow Corning</td>
			<td style="width:180px;">SYLGARD 184</td>
			<td>Silicone for dissection plates preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Dissection pins</td>
			<td style="width:288px;">Amazon</td>
			<td>B00J5PMPJA</td>
			<td>Pins for larvae positioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Tweezers</td>
			<td style="width:288px;">WPIINC</td>
			<td>500342</td>
			<td>Tweezers for placing pins, removing the guts and tracheas.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Scissors</td>
			<td style="width:288px;">WPIINC</td>
			<td>501778</td>
			<td>Scissors for cutting the cuticula of the larvae and nerves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Olympus BX61WI</td>
			<td style="width:288px;">Olympus</td>
			<td style="width:180px;">BX61WI</td>
			<td>Upright microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Olympus Lumplan FL N 60x</td>
			<td style="width:288px;">Olympus</td>
			<td style="width:180px;">UPLFLN 60X</td>
			<td>Microscope objective 60X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus UPlan FL N 10x</td>
			<td style="width:288px;">Olympus</td>
			<td style="width:180px;">Uplanfl N 10X</td>
			<td>Microscope objective 10X</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Narishige Micromanipulator</td>
			<td style="width:288px;">Narishige</td>
			<td style="width:180px;">MHW-3</td>
			<td>Three-axis Water Hydraulic Micromanipulator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">npi Electronic GmbH ELC-03XS</td>
			<td style="width:288px;">npi Electronic GmbH</td>
			<td style="width:180px;">ELC-03XS</td>
			<td>Electrophysiological amplifier</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A.M.P.I Master 8</td>
			<td style="width:288px;">A.M.P.I.</td>
			<td style="width:180px;">Master 8</td>
			<td>Electrical stimulator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A.M.P.I Iso-Flex</td>
			<td style="width:288px;">A.M.P.I.</td>
			<td style="width:180px;">Iso-Flex</td>
			<td>Stimulus isolator</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">TMC antivibration table</td>
			<td style="width:288px;">TMC</td>
			<td style="width:180px;">63-9090</td>
			<td>Antivibration table</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TMC Faraday cage</td>
			<td style="width:288px;">TMC</td>
			<td style="width:180px;">81-333-90</td>
			<td>Faraday cage</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Digidata 1322A</td>
			<td style="width:288px;">Axon Instruments</td>
			<td style="width:180px;">Digidata 1322A</td>
			<td>Digidata</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Computer</td>
			<td style="width:288px;">Dell</td>
			<td style="width:180px;">Dell Dimension 5150</td>
			<td>Computer with Win XP OS&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Electrode holder</td>
			<td style="width:288px;">WPI</td>
			<td>MEH3SW&#160;</td>
			<td style="width:495px;">Electrode holder</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Optical filter</td>
			<td style="width:288px;">Omega optical</td>
			<td style="width:180px;">XF 115-2</td>
			<td style="width:495px;">Filter cube for Green Fluorescent Protein (GFP) detection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">pCLAMP 8</td>
			<td style="width:288px;">Axon Instruments</td>
			<td style="width:180px;">8.0.0.81</td>
			<td>Software for signal recording</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Quantan</td>
			<td style="width:288px;">In-house software</td>
			<td style="width:180px;">-</td>
			<td>Software for signal processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">Canton-S (Wildtype)</td>
			<td style="width:288px;">Bloomington Stock Center</td>
			<td>64349</td>
			<td>Control fly line</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">cpx SH1</td>
			<td style="width:288px;">Generous Gift of J.T. Littleton</td>
			<td style="width:180px;">-</td>
			<td>Complexin knock-out fly line with increased spontaneous exocytosis</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:288px;height:21px;">CD8-GFP</td>
			<td style="width:288px;">Bloomington Stock Center</td>
			<td style="width:180px;">5137</td>
			<td>Fly line with neuronal fluorescent (GFP) Tag</td>
		</tr>
	</tbody>
</table>

focal macropatch recordings of synaptic currents from the drosophila larval neuromuscular junction

果蝇神经肌肉结 (NMJ) 是研究谷氨酸突触传递的一个很好的模型系统。我们描述了从可视化 boutons 在果蝇幼虫 NMJ 的突触电流的焦 macropatch 记录技术。这项技术需要定制制作记录 micropipettes, 以及一个复合显微镜, 配备了高倍率, 长距离水浸泡物镜, 差分干扰对比 (DIC) 光学, 和荧光附件.记录电极定位在选定的突触溥的顶部, 与 DIC 光学, epi 荧光, 或两者。这种技术的优点是它允许监测有限数量的释放点的突触活动。该记录电极的直径为几微米, 而位于电极外缘外的释放位置不会对记录的电流产生显著影响。记录的突触电流具有快速的动力学, 可以很容易地解决。这些优势是特别重要的研究突变飞线与增强自发或异步突触活动。

焦点 macropatch 录音可以监视所选突触 boutons 的突触活动 (图 5)。当电极位于突触溥 (图 5A, 站点 1) 的顶部时, 记录的 mEJCs (图 5C, 站点 1) 的振幅明显超过噪音水平和尖锐的上升阶段 (在毫秒范围内)。当记录电极被移离突触溥的几微米 (图 5A, 站点 2) 时, 记录的 mEJCs 的振幅几乎下降到噪音水平 (图 5B, 站点 2)。记录的 EJCs 几乎不能区别于噪音, 他们有延长的上升阶段 (图 5C, 站点2与站点 1)。
EJCs 和 mEJCs 记录的释放点数量有限, 以及局部突触电流的快速动力学, 使得突触活动的突变体能够准确检测出释放事件。这可以通过从 complexin null 突变体 (图 6A) 的 mEJCs 记录来清楚地说明。自发活动在这个突变的32中急剧升高, 因此 mEJPs 记录的细胞重叠, 不能从彼此清楚地区分 (图 6B), 而焦点录音22启用精确检测自发释放事件 (图 4A)。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56493/56493fig5.jpg"> 
图5。EJCs 和 mEJCs 的录音从选定的溥.(A) 将电极置于选定的溥 (站点 1) 上, 并将其移离溥 (站点 2)。图像显示 CD8-GFP 标记 NMJ 可视化与 epi 荧光 (A. 1), 在肌纤维 (2) 上记录电极, 并覆盖 (a. 3, a. 4), 电极位于站点 1 (a. 3) 或站点 2 (4). 缩放条 = 10 µm (B) 从溥 (站点 1) 中记录的 mEJCs 与录音噪声有明显区别, 并有快速上升的阶段。相比之下, 从现场2记录的 mEJCs 不能可靠地区别于记录噪声, 其振幅降低 several-fold, 他们有一个较慢的时效。(C) 从溥 (站点 1) 记录的 EJCs 的振幅超过了从站点2记录的 EJCs 的振幅, 并且它们的动力学速度也更快。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56493/56493fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56493/56493fig6.jpg"> 
图6。局灶 macropatch 与细胞内记录.焦点录音使 complexin 空突变体 (A) 中的 mEJCs 精确检测, 而来自此变种的胞内记录 (B) 显示出重叠且无法可靠检测的释放事件。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56493/56493fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction at JoVE.com

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

从果蝇幼虫神经肌肉交界处的突触电流的焦 Macropatch 记录

在果蝇第三龄幼虫神经肌肉交界处, 可从可视化突触 boutons 局部记录突触电流。这种技术可以监测单个突触溥的活动。

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

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Nanyang Technological University

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Neuroscience

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

6.0K 次观看.  Wayne State University. 在果蝇第三龄幼虫神经肌肉交界处, 可从可视化突触 boutons 局部记录突触电流。这种技术可以监测单个突触溥的活动。

视频: Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

在果蝇幼虫分部轴突在体内的突触小泡的可视化

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

科研

神经科学

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

在活动区域​​的解剖和影像果蝇神经肌肉交界处

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

连续光漂白划定单雪旺细胞在神经肌肉接头处

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

星形细胞转运电流派生谷氨酸间隙的时间进程与反卷积分析

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

检测<em&gt;原位</em&gt;蛋白质 - 蛋白质复合物的<em&gt;果蝇</em&gt;幼虫神经肌肉接头使用接近结扎分析

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

神经肌肉接头处:测量突触大小，碎片和改变突触蛋白密度利用共聚焦荧光显微镜

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

为什么量化事项:表型的表征在<em&gt;果蝇</em&gt;幼虫神经肌肉接头

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Here, we describe a method for loading a calcium-sensitive dye through the frog nerve stump into the nerve endings. We also present a protocol for the recording and analysis of fast calcium transients in the peripheral nerve endings.

通过神经树桩将钙染料加载到青蛙神经末梢:青蛙神经肌肉接头中的钙瞬态登记

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

测量神经肌肉接头功能

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The Drosophila larval neuromuscular junction (NMJ), a well-established model for glutamatergic synapses, has been extensively studied for decades. Identification of mutations causing NMJ morphological defects revealed a repertoire of genes that regulate synapse development and function. Many of these were identified in large-scale studies that focused on qualitative approaches to detect morphological abnormalities of the Drosophila NMJ. A drawback of qualitative analyses is that many subtle players contributing to NMJ morphology likely remain unnoticed. Whereas quantitative analyses are required to detect the subtler morphological differences, such analyses are not yet commonly performed because they are laborious. This protocol describes in detail two image analysis algorithms "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics", available as Fiji-compatible macros, for quantitative, accurate and objective morphometric analysis of the Drosophila NMJ. This methodology is developed to analyze NMJ terminals immunolabeled with the commonly used markers Dlg-1 and Brp. Additionally, its wider application to other markers such as Hrp, Csp and Syt is presented in this protocol. The macros are able to assess nine morphological NMJ features: NMJ area, NMJ perimeter, number of boutons, NMJ length, NMJ longest branch length, number of islands, number of branches, number of branching points and number of active zones in the NMJ terminal.

Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology

Two image analysis algorithms, "Drosophila NMJ Morphometrics" and "Drosophila NMJ Bouton Morphometrics" were created, to automatically quantify nine morphological features of the Drosophila neuromuscular junction (NMJ).

两种算法的高通量和多参数定量<em&gt;果蝇</em&gt;神经肌肉接头形态

Synaptic morphology is tightly related to synaptic efficacy, and in many cases morphological synapse defects ultimately lead to synaptic malfunction. The ...