Name: focal macropatch recordings of synaptic currents from the drosophila larval neuromuscular junction
Uploaded: 2017-09-25T14:00:00.000+00:00
Duration: 7 min 1 s
Description: Watch this Scientific Journal Video about Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction at JoVE.com

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/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/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/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/files/ftp_upload/56493/56493fig4large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a>

<ol>
	<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. 262 (1), 189-214 (1976).</li><li>Katz, B., Miledi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+temperature+on+the+synaptic+delay+at+the+neuromuscular+junction.">The effect of temperature on the synaptic delay at the neuromuscular junction.</a> J Physiol. 181 (3), 656-670 (1965).</li><li>Macleod, G. T., Gan, J., Bennett, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicle-associated+proteins+and+quantal+release+at+single+active+zones+of+amphibian+(Bufo+marinus)+motor-nerve+terminals.">Vesicle-associated proteins and quantal release at single active zones of amphibian (Bufo marinus) motor-nerve terminals.</a> J Neurophysiol. 82 (3), 1133-1146 (1999).</li><li>Macleod, G. T., Farnell, L., Gibson, W. G., Bennett, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantal+secretion+and+nerve-terminal+cable+properties+at+neuromuscular+junctions+in+an+amphibian+(Bufo+marinus).">Quantal secretion and nerve-terminal cable properties at neuromuscular junctions in an amphibian (Bufo marinus).</a> J Neurophysiol. 81 (3), 1135-1146 (1999).</li><li>Zefirov, A., Benish, T., Fatkullin, N., Cheranov, S., Khazipov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+active+zones.">Localization of active zones.</a> Nature. 376 (6539), 393-394 (1995).</li><li>Macleod, G. T., Lavidis, N. A., Bennett, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+dependence+of+quantal+secretion+from+visualized+sympathetic+nerve+varicosities+on+the+mouse+vas+deferens.">Calcium dependence of quantal secretion from visualized sympathetic nerve varicosities on the mouse vas deferens.</a> J Physiol. 480 (Pt 1), 61-70 (1994).</li><li>Samigullin, D., Bill, C. A., Coleman, W. L., 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=Regulation+of+transmitter+release+by+synapsin+II+in+mouse+motor+terminals.">Regulation of transmitter release by synapsin II in mouse motor terminals.</a> J Physiol. 561 (Pt 1), 149-158 (2004).</li><li>Coleman, W. L., 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=Rab3a-mediated+vesicle+recruitment+regulates+short-term+plasticity+at+the+mouse+diaphragm+synapse.">Rab3a-mediated vesicle recruitment regulates short-term plasticity at the mouse diaphragm synapse.</a> Mol Cell Neurosci. 41 (2), 286-296 (2009).</li><li>Atwood, H. L., Parnas, H., Parnas, I., Wojtowicz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantal+currents+evoked+by+graded+intracellular+depolarization+of+crayfish+motor+axon+terminals.">Quantal currents evoked by graded intracellular depolarization of crayfish motor axon terminals.</a> J Physiol. 383, 587-599 (1987).</li><li>Parnas, H., Dudel, J., Parnas, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotransmitter+release+and+its+facilitation+in+crayfish.+I.+Saturation+kinetics+of+release,+and+of+entry+and+removal+of+calcium.">Neurotransmitter release and its facilitation in crayfish. I. Saturation kinetics of release, and of entry and removal of calcium.</a> Pflugers Arch. 393 (1), 1-14 (1982).</li><li>Wojtowicz, J. M., Marin, L., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-induced+changes+in+synaptic+release+sites+at+the+crayfish+neuromuscular+junction.">Activity-induced changes in synaptic release sites at the crayfish neuromuscular junction.</a> J Neurosci. 14 (6), 3688-3703 (1994).</li><li>Zucker, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crayfish+neuromuscular+facilitation+activated+by+constant+presynaptic+action+potentials+and+depolarizing+pulses.">Crayfish neuromuscular facilitation activated by constant presynaptic action potentials and depolarizing pulses.</a> J Physiol. 241 (1), 69-89 (1974).</li><li>Zucker, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+statistics+of+transmitter+release+during+facilitation.">Changes in the statistics of transmitter release during facilitation.</a> J Physiol. 229 (3), 787-810 (1973).</li><li>Worden, M. K., Bykhovskaia, M., Hackett, 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=Facilitation+at+the+lobster+neuromuscular+junction:+a+stimulus-dependent+mobilization+model.">Facilitation at the lobster neuromuscular junction: a stimulus-dependent mobilization model.</a> J Neurophysiol. 78 (1), 417-428 (1997).</li><li>Bykhovskaia, M., Hackett, J. T., Worden, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asynchrony+of+quantal+events+in+evoked+multiquantal+responses+indicates+presynaptic+quantal+interaction.">Asynchrony of quantal events in evoked multiquantal responses indicates presynaptic quantal interaction.</a> J Neurophysiol. 81 (5), 2234-2242 (1999).</li><li>Bykhovskaia, M., Polagaeva, E., Hackett, 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=Mechnisms+underlying+different+facilitation+forms+at+the+lobster+neuromuscular+synapse.">Mechnisms underlying different facilitation forms at the lobster neuromuscular synapse.</a> Brain Res. 1019 (1-2), 10-21 (2004).</li><li>Cooper, R. L., Stewart, B. A., Wojtowicz, J. M., Wang, S., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantal+measurement+and+analysis+methods+compared+for+crayfish+and+Drosophila+neuromuscular+junctions,+and+rat+hippocampus.">Quantal measurement and analysis methods compared for crayfish and Drosophila neuromuscular junctions, and rat hippocampus.</a> J Neurosci Methods. 61 (1-2), 67-78 (1995).</li><li>Stewart, B. A., Atwood, H. L., Renger, J. J., Wang, J., 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=Improved+stability+of+Drosophila+larval+neuromuscular+preparations+in+haemolymph-like+physiological+solutions.">Improved stability of Drosophila larval neuromuscular preparations in haemolymph-like physiological solutions.</a> J Comp Physiol A. 175 (2), 179-191 (1994).</li><li>Pawlu, C., DiAntonio, A., Heckmann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postfusional+control+of+quantal+current+shape.">Postfusional control of quantal current shape.</a> Neuron. 42 (4), 607-618 (2004).</li><li>Akbergenova, Y., 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=Synapsin+maintains+the+reserve+vesicle+pool+and+spatial+segregation+of+the+recycling+pool+in+Drosophila+presynaptic+boutons.">Synapsin maintains the reserve vesicle pool and spatial segregation of the recycling pool in Drosophila presynaptic boutons.</a> Brain Res. 1178, 52-64 (2007).</li><li>Akbergenova, Y., 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=Enhancement+of+the+endosomal+endocytic+pathway+increases+quantal+size.">Enhancement of the endosomal endocytic pathway increases quantal size.</a> Mol Cell Neurosci. 40 (2), 199-206 (2009).</li><li>Vasin, A., Volfson, D., Littleton, J. T., 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=Interaction+of+the+Complexin+Accessory+Helix+with+Synaptobrevin+Regulates+Spontaneous+Fusion.">Interaction of the Complexin Accessory Helix with Synaptobrevin Regulates Spontaneous Fusion.</a> Biophys J. 111 (9), 1954-1964 (2016).</li><li>Wong, K., Karunanithi, S., Atwood, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantal+unit+populations+at+the+Drosophila+larval+neuromuscular+junction.">Quantal unit populations at the Drosophila larval neuromuscular junction.</a> J Neurophysiol. 82 (3), 1497-1511 (1999).</li><li>Dudel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+reduced+calcium+on+quantal+unit+current+and+release+at+the+crayfish+neuromuscular+junction.">The effect of reduced calcium on quantal unit current and release at the crayfish neuromuscular junction.</a> Pflugers Arch. 391 (1), 35-40 (1981).</li><li>Dudel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+Ca2++inflow+to+quantal,+phasic+transmitter+release+from+nerve+terminals+of+frog+muscle.">Contribution of Ca2+ inflow to quantal, phasic transmitter release from nerve terminals of frog muscle.</a> Pflugers Arch. 422 (2), 129-142 (1992).</li><li>Marrero, H. G., Lemos, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Loose-Patch-Clamp method. , (2007).</li><li>Wu, W. H., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+recordings+of+high+and+low+output+NMJs+on+the+crayfish+leg+extensor+muscle.">Physiological recordings of high and low output NMJs on the crayfish leg extensor muscle.</a> J Vis Exp. (45), (2010).</li><li>Verstreken, P., Ohyama, T., Bellen, H. 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><tbody><tr><td>Sutter P-97</td><td>Sutter instrument</td><td>P-97</td><td>Microelectrode puller</td></tr><tr><td>Narishige MF-830</td><td>Narishige</td><td>MF-830</td><td>Microforge</td></tr><tr><td>WPI MF200</td><td>WPI</td><td>MF200</td><td>Microforge</td></tr><tr><td>Glass capilaries</td><td>WPI</td><td>B150-86-10</td><td>Glass capilaries</td></tr><tr><td>Microtorch 1WG61</td><td>Grainer</td><td>1WG61</td><td>Microtorch</td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td>SYLGARD 184</td><td>Silicone for dissection plates preparation</td></tr><tr><td>Dissection pins</td><td>Amazon</td><td>B00J5PMPJA</td><td>Pins for larvae positioning</td></tr><tr><td>Tweezers</td><td>WPIINC</td><td>500342</td><td>Tweezers for placing pins, removing the guts and tracheas.</td></tr><tr><td>Scissors</td><td>WPIINC</td><td>501778</td><td>Scissors for cutting the cuticula of the larvae and nerves.</td></tr><tr><td>Olympus BX61WI</td><td>Olympus</td><td>BX61WI</td><td>Upright microscope</td></tr><tr><td>Olympus Lumplan FL N 60x</td><td>Olympus</td><td>UPLFLN 60X</td><td>Microscope objective 60X</td></tr><tr><td>Olympus UPlan FL N 10x</td><td>Olympus</td><td>Uplanfl N 10X</td><td>Microscope objective 10X</td></tr><tr><td>Narishige Micromanipulator</td><td>Narishige</td><td>MHW-3</td><td>Three-axis Water Hydraulic Micromanipulator</td></tr><tr><td>npi Electronic GmbH ELC-03XS</td><td>npi Electronic GmbH</td><td>ELC-03XS</td><td>Electrophysiological amplifier</td></tr><tr><td>A.M.P.I Master 8</td><td>A.M.P.I.</td><td>Master 8</td><td>Electrical stimulator</td></tr><tr><td>A.M.P.I Iso-Flex</td><td>A.M.P.I.</td><td>Iso-Flex</td><td>Stimulus isolator</td></tr><tr><td>TMC antivibration table</td><td>TMC</td><td>63-9090</td><td>Antivibration table</td></tr><tr><td>TMC Faraday cage</td><td>TMC</td><td>81-333-90</td><td>Faraday cage</td></tr><tr><td>Digidata 1322A</td><td>Axon Instruments</td><td>Digidata 1322A</td><td>Digidata</td></tr><tr><td>Computer</td><td>Dell</td><td>Dell Dimension 5150</td><td>Computer with Win XP OS</td></tr><tr><td>Electrode holder</td><td>WPI</td><td>MEH3SW</td><td>Electrode holder</td></tr><tr><td>Optical filter</td><td>Omega optical</td><td>XF 115-2</td><td>Filter cube for Green Fluorescent Protein (GFP) detection</td></tr><tr><td>pCLAMP 8</td><td>Axon Instruments</td><td>8.0.0.81</td><td>Software for signal recording</td></tr><tr><td>Quantan</td><td>In-house software</td><td>-</td><td>Software for signal processing</td></tr><tr><td>Canton-S (Wildtype)</td><td>Bloomington Stock Center</td><td>64349</td><td>Control fly line</td></tr><tr><td>cpx SH1</td><td>Generous Gift of J.T. Littleton</td><td>-</td><td>Complexin knock-out fly line with increased spontaneous exocytosis</td></tr><tr><td>CD8-GFP</td><td>Bloomington Stock Center</td><td>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/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/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 ...

focal-macropatch-recordings-synaptic-currents-from-drosophila-larval

Nanyang Technological University

Research

JoVE Journal

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

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

配对补丁钳从电机神经元和斑马鱼的目标骨骼肌

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

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 ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

从查明的神经元和取食行为的钙信号的同时记录果蝇</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

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. ...

生物学

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 ...

Preparation of Drosophila Central Neurons for in situ Patch Clamping

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker1,2. Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain3,4 and ventral nerve cord of embryonic5,6, larval7,8,9,10, and adult Drosophila11,12,13,14. A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN515), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.

Preparation of Drosophila Central Neurons for in situ Patch Clamping

In situ patch clamp recordings are used for electrophysiological characterization of neurons in intact circuitry. In the Drosophila genetic model patch clamping is difficult because the CNS is small and surrounded by a robust sheath. This article describes the procedure to remove the sheath and clean neurons for subsequent patch clamp recordings.

制备<em&gt;果蝇</em&gt;中央神经元<em在原位</em&gt;膜片钳

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience ...

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

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

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

从胚胎斑马鱼的毛特纳细胞膜片钳记录

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at the Drosophila NMJ enables researchers to acquire synaptic information, encompassing both quantitative data on synapse abundance and detailed insights into their morphology. However, the diffuse distribution and limited visual range of the TEM present challenges for the ultrastructural analysis. This study introduces an innovative and efficient sample preparation method that surpasses the conventional approach. The procedure begins by placing a metal mesh at the base of a flat-bottomed bottle or test tube, followed by positioning fixed larvae samples onto the mesh. An additional mesh is placed over the samples, ensuring that they are positioned between the two meshes. The fixed samples are thoroughly dehydrated and infiltrated before proceeding with the embedding procedure. Then embedding of the samples in epoxy resin is performed in a flat sheet manner, which allows for the preparation of muscles for positioning and sectioning. Benefiting from these steps, all the muscles of Drosophila larvae can be visualized under light microscopy, therey facilitating subsequent positioning and sectioning. Excess resin is removed after locating the 6th and 7th muscles of body segments A2 and A3. Serial ultra-thin sectioning of the 6th or 7th muscle is performed.

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

This report provides a new sample preparation procedure for visualizing neuromuscular junctions in Drosophila Larvae. This method is more effective in preventing the curling of the samples compared to the traditional method and is particularly useful for Drosophila neuromuscular junction ultrastructural analysis.

优化 果蝇 幼虫神经肌肉接头透射电子显微镜样品制备工艺

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at ...

Drosophila Neuromuscular Junction (NMJ) Quantification: A Method to Assess Synaptic Morphology and Function

Source: Castells-Nobau, A., et al. <a href="https://www.jove.com/video/55395/" target="_blank">Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology</a>. J. Vis. Exp. (2017).
The Drosophila neuromuscular junction (NMJ) is used as a model to study the morphology and function of synapses. This video describes important features that researchers quantify to characterize the NMJ. The example protocol demonstrates a software able to perform the quantification automatically.

Drosophila Neuromuscular Junction NMJ Quantification: A Method to Assess Synaptic Morphology and Function

果蝇神经肌肉接头 （NMJ） 定量:一种评估突触形态和功能的方法

Source: Castells-Nobau, A., et al. Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology. ...

从果蝇幼虫神经肌肉交界处的突触电流的焦 Macropatch 记录

摘要

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此视频中的章节

配对补丁钳从电机神经元和斑马鱼的目标骨骼肌

在活动区域的解剖和影像果蝇神经肌肉交界处

从查明的神经元和取食行为的钙信号的同时记录果蝇

电生理方法记录从果蝇幼虫NMJ突触潜力

Channelrhodopsin2介导的突触电位刺激果蝇神经肌肉接头

制备

在电生理记录果蝇胚胎

从胚胎斑马鱼的毛特纳细胞膜片钳记录

优化果蝇幼虫神经肌肉接头透射电子显微镜样品制备工艺

果蝇神经肌肉接头（NMJ）定量:一种评估突触形态和功能的方法

从果蝇幼虫神经肌肉交界处的突触电流的焦 Macropatch 记录

摘要

探索更多视频

此视频中的章节

配对补丁钳从电机神经元和斑马鱼的目标骨骼肌

在活动区域​​的解剖和影像果蝇神经肌肉交界处

从查明的神经元和取食行为的钙信号的同时记录果蝇

电生理方法记录从果蝇幼虫NMJ突触潜力

Channelrhodopsin2介导的突触电位刺激果蝇神经肌肉接头

制备

在电生理记录果蝇胚胎

从胚胎斑马鱼的毛特纳细胞膜片钳记录

优化 果蝇 幼虫神经肌肉接头透射电子显微镜样品制备工艺

果蝇神经肌肉接头 （NMJ） 定量:一种评估突触形态和功能的方法

在活动区域的解剖和影像果蝇神经肌肉交界处

优化果蝇幼虫神经肌肉接头透射电子显微镜样品制备工艺

果蝇神经肌肉接头（NMJ）定量:一种评估突触形态和功能的方法