Name: acute dissociation of lamprey reticulospinal axons to enable recording from the release face membrane of individual functional presynaptic terminals
Uploaded: 2014-10-01T14:00:00.000+00:00
Duration: 12 min 1 s
Description: Watch this Scientific Journal Video about Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals at JoVE.c

This work has been supported by NINDS, RO1NS52699 and MH84874 to SA. 
We would like to thank Dr. Dave Featherstone (Department of Biological Sciences, University for Illinois at Chicago) for providing us with the suture glue used in the immunohistochemistry work. We thank Michael Alpert for his comments and proofreading of the manuscript.

1，制备聚-D-赖氨酸氢溴酸<ol><li>制备1毫克/毫升的聚-D-赖氨酸氢溴酸盐在0.1M硼酸盐缓冲液（pH 8.5）。 </li><li>分装和储存在-20℃。 </li></ol>盖玻片的二聚赖氨酸涂层注意:进行在层流室中所有的清洗和涂层的步骤。 <ol><li>放置盖玻片含1N盐酸（HCl）的2小时培养皿。 </li><li>吸出所有的HCl冲洗，用70％乙醇（EtOH中）2-3倍。 </li><li>发表在70％EtOH中搅拌1小时。 </li><li>吸出的所有70％乙醇漂洗用100％的乙醇2-3倍。 </li><li>留在100％EtOH中搅拌2小时。吸出的所有乙醇。 </li><li>甩干用滤纸和空气干燥几秒。 </li><li>地方的O / N合1毫克/毫升多聚赖氨酸的解决方案。 </li><li>冲洗盖玻片第二天用微孔水4-5倍。 </li><li>空气干燥多聚赖氨酸涂在玻璃辊盖玻片在一个干净的培养皿中。 </li><li>对于immunohistochemistrY，使用35×10毫米的培养皿的盖子。放入盘中，以厚度为0.3厘米，允许设置在30℃的CO / N:准备SYLGARD（polydimethylsilioxane，PDMS）浇注PDMS（按重量计1弹性体和固化剂10）衬里的菜肴。一旦树立，1厘米嵌入到PDMS的切2厘米。用聚赖氨酸按照相同的步骤与上述用于盖玻片清洁和涂层的插图。 </li><li>商店聚赖氨酸包被的盖玻片上，并菜肴覆盖层流室中，直到使用（不超过2周，但通过周期性地制备每3-4天达到最好的粘合剂的结果）。 </li><li>准备一个硅橡胶块，引脚脊髓中的振动组织切片机。混弹性体A和弹性体B 1:1（重量）。倒入100×15毫米培养皿中，并允许设置在o / n 30℃。剪下一块长方形的PMDS和使用环氧树脂胶是胶水切片底板。让胶水来设置固定到位的PDMS和切片多个薄切片切断面，以获得平坦表面到管脚上的组织。 </li></ol>对七鳃鳗脊髓3。急性解离的收益率隔离网状脊髓轴突<ol><li>麻醉的ammoecoete或成年鳗（Petromyzon马里努斯 ）与三卡因甲磺酸盐（MS-222; 100毫克/升）。添加麻醉剂入水，在塑料杯中覆盖了盖子，包含七鳃鳗被牺牲。 </li><li>斩首鳗中冷（4℃）的组成如下（以mM计）的林格氏溶液:130的NaCl，2.1氯化钾，2.6的CaCl 2，1.8的MgCl 2，4 HEPES，4葡萄糖（pH为7.6，渗透压270毫渗透摩尔浓度）和去除体墙上的肌肉暴露脊髓背侧面。 </li><li>从用细镊子脊髓的背侧面删除脑膜野蛮。请不要在此阶段移除腹脑膜野蛮。 </li><li>切断脊髓1厘米长的小段。 </li><li>用细昆虫针，背一面朝上针脊髓片，在硅橡胶内衬切片解放军基地TE（见2.12）中含有冰冷的林格氏液振动组织切片机室。 </li><li>由沿与所述叶片的rostro-尾轴切片取出脊髓背柱的中央部分，留下的喙和尾部的完整背柱段结束服务的解离过程图2a和图2b中的处理。用最慢的速度设定，允许分片，并且只删除背柱下一层网状脊髓轴突柱完好无损图2b的深度设置。 </li><li>在1毫克/毫升蛋白酶（从灰色链霉菌种类十四）和1毫克/毫升胶原酶林格氏液制备（IA型溶组织梭菌）鸡尾酒孵育切片脊髓片在室温下45分钟（改编自萨尔瓦多Manira＆Bussières ，1997 17）。 </li><li>用细销的PDMS销酶处理脊髓件，内衬培养皿CONTA进不去冷的（4℃），林格氏溶液。 </li><li>用细镊子取出腹脑膜野蛮。 </li><li>切断脊髓 ​​侧束用解剖刀刀片的切片背侧部离开网状脊髓轴突完好脊髓图2d的中央柱的中点。 </li><li>将浸油的镜头上的下降。 </li><li>将聚赖氨酸包被的盖玻片（ 图3a，顶片，红色矩形）中的录音室图3中的槽和在图3a中应用真空润滑脂的所有边缘（空间红色和蓝色矩形之间，以促进密封。螺丝顶到位。将室中录制钻机上的插图。 </li><li>添加林格氏溶液成使用巴氏吸管录音室。 </li><li>连接含有防冻剂溶液的热电式冷却装置的输入管道中的压力瓶的流出管道。连接OUTPUT管的热电式冷却装置的外冷却夹套的输入端和输出端连接到贮存器图3b中。 </li><li>将一块脊髓在录音室的时候，轻轻分开脊髓沿盖玻片在使用特富龙涂层的镊子始终保持它，直到轴突隔离图2e和图2f上 。 </li><li>通过使加压（氮气压入瓶）防冻剂溶液（位移），通过对热电冷却装置中，通过记录室图3b的外冷却夹套将记录溶液温度至10℃。 </li><li>允许轴突至在10℃下恢复1小时后分解。 </li></ol><img alt="图2" fo:content-width="4in" src="/files/ftp_upload/51925/51925fig2highres.jpg" width="400" /> <strong&gt;图2原理分离协议网状脊髓轴突隔离的说明。（一）拆除背柱。箭头指示的组织切片的方向。在背角的由字母D（红色字体颜色）标记，而腹角由字母V（红色字体颜色）（B）背柱脊髓露出网状脊髓轴突的中心部分除去（绿线）。腹侧角，其中该限幅处理后保持不动，被标记的字母V（红色字体颜色）（C）45分钟治疗用蛋白酶和胶原酶酶鸡尾酒（1毫克/毫升）。横向束（四）切割脊髓;指示位置，方向和横向切割的程度。（五）脊髓的机械分解。箭头表示钳子和离解过程中的分离力方向的位置（F）Representati例如已经分离网状脊髓轴突的准备。绿色箭头标记的急性分离网状脊髓轴突地区没有任何突触后处理。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51925/51925fig3highres.jpg" width="500" /> 提供图3示意录音室电实验。尺寸为英寸。在basepiece图（A）所示的红色矩形表示盖玻片在basepiece盖玻片槽定位。红色和蓝色矩形之间的区域中，在basepiece图（A）所示为其中的高真空润滑脂被施加（b）表示组装的录音室中。 4，标签和突触前终端的识别与FM 1-43 <ol><li>标签突触前终端，通过将FM 1-43为V期间，在高K +去极化突触外切内吞作用esicles。灌注制备具有5微米调频1-43（在含有30 mM的氯化钾5毫升林格氏溶液）。 </li><li>灌注制备用1毫克/毫升Advasep-7（5毫升林格氏溶液）以除去过量的调频染料）18。 </li><li>灌注制剂与林格氏溶液15分钟，洗出任何remant染料和Advasep。 </li><li>图像与100×油镜（NA 1.25）的倒置荧光显微镜，在用数字CCD相机和图像采集软件事无巨细结合，使用标准荧光成像协议。 </li><li>确定荧光标记的突触前终端为目标的记录图4c和图4d。 </li></ol> 5，免疫隔离网状脊髓轴突<ol><li>填写菜插图（协议第2.10）与二价离子（ 钙离子和镁离子 ）免费林格氏溶液。 </li><li>制作补丁移液器在P-87微量拉马。将少量的缝线胶在使用补丁吸管（1.5毫米外径的玻璃）和吸入（使用硅胶管0.89毫米内径与连接到吸入端的微量移液器尖端）的多聚赖氨酸插入的一端。 </li><li>进行使用相同的程序解离中提到的协议部3 如图2所示 。放置在缝合胶水，并轻轻按下用镊子脊髓的一端牢固地附着到表面上。放置另一个一滴缝合胶水在插块的另一端，并轻轻拉伸脊髓直到轴突的解离以及通过轻轻拖曳它在缝合胶水附着自由脊髓末端。轻轻向下按压的钳固定到位。 </li><li>交换价释放林格定期林格氏液灌注液。 </li><li>允许轴突恢复20分钟后dissociation在10℃下。 </li><li>固定游离轴突在4％多聚甲醛（PFA）进行20分钟{制备磷酸盐缓冲盐水（PBS，（毫摩尔）NaCl的137，氯化钾2.7，的Na 2 HPO 4 10 KH 2 PO 4 1.8，pH值7.4）}。过滤前的PFA溶液使其通过0.2微米针筒过滤器来使用。 </li><li>通过灌注0.1M甘氨酸（在PBS）10分钟洗出的煤灰。 </li><li> 10分钟温育在0.1％的Triton-X（在PBS中）。 </li><li>由PBS灌注20分钟洗净。 </li><li>用5％无脂牛奶（在PBS中）6小时，在4℃阻断。 </li><li>加于初级抗体对感兴趣VGCC（1:200稀释于PBS中），并孵育20小时，在4℃。 </li><li>由PBS灌注20分钟洗净。 </li><li>用5％无脂牛奶（在PBS中），在4℃下10分钟的阻断。 </li><li>加在第二抗体（1:400稀释于PBS中），并培育在黑暗2小时，在4℃。 </li><li>用PBS灌注20分钟洗净。 </li><li>用1％的牛血清Albu阻止分（在PBS中），20分钟。 </li><li>在添加的Alexa Fluor 488鬼笔环肽（5个/μL工作浓度;股票在甲醇200单位/毫升的准备）。 </li><li>用PBS灌注20分钟洗净。 </li><li>图片采用100X水浸泡镜片上的共聚焦显微镜。 </li></ol> 6，电生理记录<ol><li>在P-87微量拉马制造铝硅酸盐玻璃修补移液器（移液管电阻2-5MΩ）。设计补丁吸管，这样枪头涵盖整个突触前终端直径。 </li><li> PDMS涂层补丁移液器通过浸渍在50〜60 psi的压力的膜片吸管插入PDMS 23（附加一个硅胶管，0.89毫米内径，连接到氮气气缸，该补丁移液管的后端）和干燥用热枪。交替地，以手动方式涂敷移液管与PDMS，施加涂层尽可能靠近尖端尽可能的化合物，显微镜下观察。 </li><li>使用MICR消防波兰补丁移液器oforge（一定制的铂丝装到一个复合式显微镜的阶段）。 </li><li>确定分离的轴突通过荧光显微镜证实标记的突触前末梢。 </li><li>填写记录溶液（旨在隔离的 Ca 2 +电流;的10mM的CaCl 2或90 mM的氯化钡2作为电荷载体，HEPES缓冲的pH值7.6，渗透压270毫渗透摩尔浓度）到使用注射器的补丁吸管。 </li><li>将补丁吸管插入使用机动操纵器MP225移液器支架和位置上面洗澡。轻轻放下补丁吸管放入浴缸和位置对一个荧光标识突触前终端的脸。 </li><li>推进补丁吸管缓慢，直到接触是用膜。在这一点上，通过安装于移液管保持器的管实现千兆欧姆密封由平缓口吸入。 </li><li>为了达到要求的记录单声道钙电流极低的背景噪音水平，使用Axopatch 200B与冷却探头的录像。在20-50 kHz和过滤器使用5 kHz的贝塞尔滤波器的样本数据。开展利用Axograph十，数据采集</li><li>使用标准的STEP协议，以10 mV递增的刺激。集成了预脉冲的协议，该协议的步骤之前，确保钙离子通道的最大激活。集成了10 mV的步泄漏到泄漏电流的分析后减的步骤协议。 </li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+analysis+of+synaptic+vesicle+recycling+at+the+frog+neuromuscular+junction.">Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction.</a> Science. 255 (5041), 200-203 (1992).</li><li>Bleckert, A., Photowala, H., Alford, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+pools+of+actin+at+presynaptic+terminals.">Dual pools of actin at presynaptic terminals.</a> Journal of Neurophysiology. 107 (12), 3479-3492 (2012).</li><li>Gustafsson, J. S., Birinyi, A., Crum, J., Ellisman, M., Brodin, L., Shupliakov, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+organization+of+lamprey+reticulospinal+synapses+in+three+dimensions.">Ultrastructural organization of lamprey reticulospinal synapses in three dimensions.</a> The Journal of Comparative Neurology. 450 (2), 167-182 (2002).</li><li>Broadie, K., Featherstone, D. E., Chen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+Recording+in+the+Drosophila+Embryo.">Electrophysiological Recording in the Drosophila Embryo.</a> Journal of Visualized Experiments. (27), (2009).</li><li>Rae, J. L., Levis, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+exceptionally+low+noise+single+channel+recordings.">A method for exceptionally low noise single channel recordings.</a> Pfl&#252;gers Archiv European Journal of Physiology. 420 (5-6), 618-620 (1992).</li></ol>

The authors do not have any competing financial interests or other conflicts of interest to disclose.

我们离解协议是由产生隔离网状脊髓轴突缺乏突触后预测图2F显著，但仍然保留功能性突触前终端图4c和图4d。如果没有突触后处理反对突触前终端允许在单个突触前终端直接记录进入突触前释放的脸膜，以前不可能在中央和突触只有两个肾盏型突触前终端成功实现;小鸡睫状神经节7-10和大鼠肾盏11,12的花萼型突触。个别的突触前末梢，其中在?...

突触传递是极其快速和精确的过程。动作电位在突触前终端的入侵导致了开放位于剥离面的膜，由此带来的突触前钙作为触发的囊泡融合和神经递质的释放1 VGCCs的。所有这些步骤发生几百微秒2的范围内，并因此需要VGCCs的囊泡融合机械3的紧空间耦合。突触前钙离子通量一直主要通过影像学方法，使用钙离子敏感的染料4特点。结合的 Ca 2 +的缓冲液调制的 Ca 2 +在突触前神经元已被用于间接地表征突触前钙和神经传递3之间的关系。另外，通过解笼锁的 Ca 2 + 5或记录macroscopi调节突触前游离的 Ca 2 +浓度Ç 的 Ca 2 +电流，一直配合使用囊泡融合和/或释放的措施;如电容测量6或突触后反应2，解决同样的问题。然而， 离子电流直接剥离面表征钙，突触前膜的特殊部分，其中膜去极化转化为钙离子电流，引发突触囊泡融合和神经递质的释放，是不可或缺的获得钙的精确测量离子要求突触囊泡融合。此外，能够直接在个人突触前末梢表征的 Ca 2 +电流，加上囊泡融合和释放的精确同步测量允许的动作电位时程之间的定时关系的精确澄清，突触前的 Ca 2 +电流，囊泡融合和释放。访问该释放面膜是不是在大多数由于通过突触后树突关闭并置的突触前末梢的提供。这交通不便一直在VGCCs的特性的一大障碍，因为它可以防止电流的个体突触前终端的直接测量。突触前钙电流个别突触前终端的直接表征迄今未能在中央突触，并只有两个肾盏型突触前终端已经实现;小鸡睫状神经节7-10和大鼠肾盏11,12的花萼型突触。在所有其它突触前的终端包括七鳃鳗脊髓13巨网状脊髓突触，缺乏进入突触前释放的脸膜因此有必要采用间接的方法，如钙成像研究突触前钙离子通量。 <img alt="图1" f○:内容宽度="5英寸"SRC ="/文件/ ftp_upload / 51925 / 51925fig1highres.jpg"WIDTH ="500"/&gt; 图1:七鳃鳗巨型网状脊髓突触。（一）横截面七鳃鳗脊髓表明背腹方向。网状脊髓轴突都标有绿色星号（B）3-D中的网状脊髓突触中的七鳃鳗脊髓显示突触前网状脊髓轴突使得无数顺便接点（标记绿色箭头）到突触后神经元13的重建。突触前终端已经被标记的Alexa Fluor 488的酰肼轭毒伞素（绿色），而突触后神经元已经充满的Alexa Fluor 568的酰肼（红色）。 鳗巨型网状脊髓轴突，位于平行脊髓延髓-尾轴图1a，形式多种顺便突触联系的腹侧区域上的神经元脊髓前角14 图1b 13。在完整的脊髓13,15宏观全细胞钙电流已经记录从网状脊髓轴突。然而，以往的盲目尝试直接测量的Ca 2 +电流在用细胞贴附膜片钳技术的完整的七鳃鳗脊髓网状脊髓轴突已经证明不成功的13由于缺乏进入突触前释放的脸膜由于反对突触后处理图1b。剥离面膜已经预先，记 ​​录12或酶处理加上机械解离16前突触的机械扰动进行访问除去突触后神经元11。给出的脊髓的复合组织，其将被证明非常难以识别突触后神经元上和机械缩回它​​或扰乱日ê突触。因此，我们决定用酶处理17接着机械解离。 使用这种方法，我们已经开发了一种急性分离的七鳃鳗脊髓能够产生可行的隔离网状脊髓轴突与功能性的突触前终端没有任何突触后过程的准备，从而提供不受限制的访问单个突触前终端。在一个标准的倒置显微镜和荧光成像相结合，它使我们能够识别并锁定个人荧光鉴定突触前末梢，用补丁吸管含录音解决方案，隔离的 Ca 2 +电流图4c和图4d，使用小区记录附加电压钳技术。 钙离子电流已在个别突触前末梢图4F突触前释放的脸膜直接记录。这是一个标志意义NT突破突触传递的领域，因为它是第一个这样的记录来进行，在中央突触。

<table><tbody><tr><td>0.2 &amp;micro;m Syringe filter</td><td>EMD Millipore</td><td>SLGV004SL</td><td></td></tr><tr><td>22 x 60 mm Coverslips</td><td>Fisherbrand</td><td>12545J</td><td></td></tr><tr><td>Advasep-7</td><td>Cydex Pharmaceuticals</td><td>ADV7</td><td></td></tr><tr><td>Alexa Fluor 488 Phalloidin</td><td>Invitrogen/Life Technologies</td><td>A12379</td><td></td></tr><tr><td>Alexa-633 conjugated goat anti-rabbit secondary antibody</td><td>Invitrogen/Life Technologies</td><td>A21070</td><td></td></tr><tr><td>Antifreeze</td><td>Prestone</td><td></td><td></td></tr><tr><td>Boric acid</td><td>Sigma-Aldrich</td><td>B7660</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>Bright field light source</td><td>Dolan-Jenner</td><td>Fiberlite 180</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma Aldrich</td><td>C4901</td><td></td></tr><tr><td>Collagenase Type IA from Cloristridium Histolyticum</td><td>Sigma-Aldrich</td><td>C9891</td><td></td></tr><tr><td>Cover slip</td><td>Fisher-Scientific</td><td>12-545-J</td><td></td></tr><tr><td>Dextrose</td><td>Sigma-Aldrich</td><td>D9559</td><td></td></tr><tr><td>Digital CCD Camera</td><td>Hamamatsu</td><td>C8484-03G01</td><td></td></tr><tr><td>Dissection fine forceps</td><td>Fine Science Tools</td><td>91150-20</td><td></td></tr><tr><td>Dissection forceps</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Dissection microscope</td><td>Leica Biosystems</td><td>Leica MZ 12</td><td></td></tr><tr><td>Dissection scissors</td><td>Fine Science Tools</td><td>15025-10</td><td></td></tr><tr><td>Dissection scissors fine</td><td>Fine Science Tools</td><td>91500-09</td><td></td></tr><tr><td>Dissection scissors ultra fine</td><td>Fine Science Tools</td><td>15000-08</td><td></td></tr><tr><td>FM 1-43</td><td>Invitrogen/Life Technologies</td><td>T3163</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G7126</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H7523</td><td></td></tr><tr><td>High vacuum grease</td><td>Dow-Corning</td><td></td><td></td></tr><tr><td>Hydrochloric acid</td><td>Fisherbrand</td><td>SA-56-500</td><td></td></tr><tr><td>Immersion oil</td><td>Fisher-Scientific</td><td>M2000</td><td></td></tr><tr><td>Industrial grade nitrogen gas tank</td><td>Praxair</td><td>UN1066</td><td></td></tr><tr><td>Insect pins</td><td>Fine Science Tools</td><td>26002-10</td><td></td></tr><tr><td>Liquid suture glue</td><td>Braun Veterinary Cair Division</td><td>8V0305</td><td>The suture glue we used in our experiments was provided to us by another lab. It is no longer manufactured. We have sourced a Histoacryl Suture Glue for future use from Aesculap (Ts1050071FP)</td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M2670</td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>154903</td><td></td></tr><tr><td>Non-fat dry milk</td><td>Cell Signaling Technology</td><td>9999S</td><td></td></tr><tr><td>P-87 Micropipette puller</td><td>Sutter Instruments</td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>Perfusion pump</td><td>Cole-Palmer</td><td>Masterflex C/L</td><td></td></tr><tr><td>Petri dish 100 x 15 mm</td><td>Fisher-Scientific</td><td>875712</td><td></td></tr><tr><td>Petri dish 35 x 10 mm</td><td>Fisher-scientific</td><td>875712</td><td></td></tr><tr><td>Poly-D-lysine hydrobromide</td><td>Sigma-Aldrich</td><td>P1024</td><td>MW &gt; 300,000</td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma-Aldrich</td><td>P5379</td><td></td></tr><tr><td>Primary antibodies R-type calcium channel</td><td>Alomone Labs</td><td>ACC-006</td><td></td></tr><tr><td>Protease Type XIV from Streptomyces Griseus</td><td>Sigma-Aldrich</td><td>P5147</td><td></td></tr><tr><td>Scalpel blades</td><td>World Precision Instruments&amp;nbsp;</td><td>500240</td><td></td></tr><tr><td>Schot Duran Pressure Bottle</td><td>Fisher-Scientific</td><td>09-841-006</td><td></td></tr><tr><td>Silicone tubing for glue application</td><td>Cole-Palmer</td><td>07625-26</td><td></td></tr><tr><td>Slicing base plate</td><td>Leica Biosystems</td><td>14046327404</td><td></td></tr><tr><td>Slicing chamber</td><td>Leica Biosystems</td><td>14046230132</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>Sodium hydroxide</td><td></td><td>S8045</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma-Aldrich</td><td>S9763</td><td></td></tr><tr><td>Sodium tetraborate</td><td>Sigma-Aldrich</td><td>B3545</td><td></td></tr><tr><td>Sylgard 160 Silicone Elastomer Kit</td><td>Dow Corning&amp;nbsp;</td><td>SYLGARD&amp;reg; 160</td><td>To prepare, mix elastomer A and elastomer B 10:1 by weight</td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td>SYLGARD&amp;reg; 184&amp;nbsp;</td><td>To prepare, mix elastomer and curing agent 10:1 by weight</td></tr><tr><td>Teflon coated forceps</td><td>Fine Science Tools</td><td>11626-11</td><td></td></tr><tr><td>Tricaine methanesulphonate</td><td>Sigma-Aldrich</td><td>A5040</td><td></td></tr><tr><td>Vibratome blades</td><td>World Precision Instruments&amp;nbsp;</td><td>BLADES</td><td></td></tr><tr><td>Xenon lamp</td><td>Nikon</td><td></td><td></td></tr></tbody></table>

acute dissociation of lamprey reticulospinal axons to enable recording from the release face membrane of individual functional presynaptic terminals

突触传递是极其快速的过程。动作电位驱动的 Ca 2 +的内流进入突触前末梢，通过设在剥离面的膜电压门控性钙通道（VGCCs），是在触发小泡的融合和神经递质的释放。至关重要的突触传递的快速性的动作电位，VGCCs的到来和神经递质的释放机制之间的空间和时间同步。直接记录的Ca从个体的突触前末梢的释放面膜2 +电流的能力是必要的突触前的 Ca 2 +和神经递质的释放之间的关系的精确了解。进入突触前释放的脸膜电生理记录是不是在大多数准备，突触前钙离子可进入已使用的成像技术和宏观电流measureme特点NTS -没有足够的时间分辨率，以可视化的 Ca 2 +进入的技术。 VGCCs直接在单个突触前终端的特征已经不可能在中央突触和迄今一直只在鸡睫状神经节的花萼型突触，并在大鼠肾盏成功实现。我们已经成功地在七鳃鳗脊髓通过制定急性分离脊髓能够产生孤立的网状脊髓轴突与功能性的突触前终端缺乏突触后结构的准备的巨型网状脊髓突触解决了这个问题。我们可以荧光标记和识别单个突触前终端和针对他们的记录。使用这种准备，我们直接采用免疫组化和电生理的方法个别突触前末梢释放的脸表征VGCCs。 钙离子电流一直在剥离面的膜Ø直接记录˚F单个突触前末梢，第一个这样的记录来进行在中央突触。

<html>这离解协议产量的健康和功能性孤立的网状脊髓轴突缺乏突触后预测图2F，但仍然能够保持诱发的突触囊泡外切和内吞图4c和图4d功能性突触前终端。在网状脊髓轴突隔离区的部分可以在光学显微镜下可以清楚地识别要明确任何其他神经元突起允许无限制地访问网状脊髓轴突膜图2f的。轴突保留解离后的结构完整性。分离的轴突修补在全细胞构...

Watch this Scientific Journal Video about Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals at JoVE.c

Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals.

七鳃鳗网状脊髓轴突急性分解到从单个功能突触前终端剥离面膜启用记录

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated ...

acute-dissociation-lamprey-reticulospinal-axons-to-enable-recording

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

8.7K 次观看.  University of Illinois at Chicago. Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals. 

视频: Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

Physiological Recordings of High and Low Output NMJs on the Crayfish Leg Extensor Muscle

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor neurons innervating the extensor muscle in the walking leg of a crayfish. Distinct differences are present in the physiology and morphology of the phasic and tonic nerve terminals. The tonic axon contains many more mitochondria, enabling it to take a vital stain more intensely than the phasic axon. The tonic terminals have varicosities, and the phasic terminal is filiform. The tonic terminals are low in synaptic efficacy but show dramatic facilitated responses. In contrast, the phasic terminals are high in quantal efficacy but show synaptic depression with high frequency stimulation. The quantal output is measured with a focal macropatch electrode placed directly over the visualized nerve terminals. Both phasic and tonic terminals innervate the same muscle fibers, which suggests that inherent differences in the neurons, rather than differential retrograde feedback from the muscle, account for the morphological and physiological differentiation.

This article demonstrates how to conduct electrophysiological recordings of synaptic responses on the extensor muscle in the walking leg of a crayfish and how the nerve terminals are visualized to show the gross morphological differences of high- and low-output nerve terminals.

小龙虾腿伸肌高和低输出NMJs生理录音

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor ...

科研

神经科学

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

全细胞记录的光在视网膜切片诱发兴奋性突触电流

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

生物学

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

膜片钳电容测量和Ca 2 +单神经末梢成像在视网膜切片

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

完全性脊髓损伤和脑解剖协议后续Wholemount<em&gt;原位</em&gt;杂交苗种海七鳃鳗

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

记录光诱发神经元突触后反应在暗适应，小鼠视网膜切片准备利用膜片钳技术

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The technique is very cost-effective and easily undertaken with materials commonly found in most electrophysiology laboratories, or easily purchased. The dissection is simple and fast, with the mechanical displacement provided by a generic electroceramic wafer controlled by proprietary software. The same software also records and analyses the electroneurogram output. The recording of the evoked nerve activity is through a commercial differential amplifier connected to fire-polished standard glass microelectrodes. Helpful tips are given for improving the quality of the preparation, the stimulation and the recording conditions to optimize recording quality. The system is suitable for assaying the electrophysiological and optical properties of lanceolate terminals of palisade endings of hair follicles, as well as the outcomes from their pharmacological and/or genetic manipulation. An example of combining electrical recording with mechanical stimulation and labeling with a styryl pyridinium vital dye is given.

A simple and novel technique for recording afferent discharge due to mechanical stimulation of lanceolate terminals of palisade endings innervating mouse ear skin hair follicles is presented.

机械刺激传入输出和神经末梢标签联合录制在小鼠毛囊披针形结局

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

爪蟾Tectal 神经元全细胞膜片钳记录的制备与协议

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

Live Imaging of Synaptic Vesicle Recycling in the Neuromuscular Junction of Dissected Larval Zebrafish

Neuronal communication is mediated by synaptic transmission, which depends primarily on the release of neurotransmitters stored in synaptic vesicles (SVs) in response to an action potential (AP). Since SVs are recycled locally at the presynaptic terminal, coordination of SV exocytosis and endocytosis is important for sustained synaptic transmission. A pH-sensitive green fluorescent protein, called pHluorin, provides a powerful tool to monitor SV exo/endocytosis by targeting it to the SV lumen. However, tracking AP-driven SV recycling with the pHluorin-based probes is still largely limited to in vitro culture preparations because the introduction of genetically encoded probes and subsequent optical imaging is technically challenging in general for in vivo animal models or tissue preparations. Zebrafish is a model system offering valuable features, including ease of genetic manipulation, optical clarity, and rapid external development. We recently generated a transgenic zebrafish that highly expresses a pHluorin-labeled probe at motor neuron terminals and developed a protocol to monitor AP-driven SV exo/endocytosis at the neuromuscular junction (NMJ), a well-established synapse model that forms in vivo. In this article, we show how to prepare larval zebrafish NMJ preparation suitable for pHluorin imaging. We also show that the preparation allows time-lapse imaging under conventional upright epifluorescence microscope, providing a cost-effective platform for analyzing NMJ function.

The zebrafish larval neuromuscular junction is an attractive model for studying synaptic physiology. It is amenable to many experimental techniques, including electrophysiology and optical imaging. Here, we describe a protocol for imaging synaptic transmission using a pHluorin-based probe under an upright epifluorescence microscope.

解剖斑马鱼幼鱼神经肌肉接头突触囊泡循环的实时成像

Neuronal communication is mediated by synaptic transmission, which depends primarily on the release of neurotransmitters stored in synaptic vesicles (SVs) ...

Light-Evoked Postsynaptic Responses in Neurons of Mouse Retinal Slices

Source: Hellmer, C. B., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/52422">Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques.</a> J. Vis. Exp. (2015).
This video demonstrates the recording of light-evoked post-synaptic responses in a dark-adapted mouse retinal tissue slice preparation. The retinal slice is placed in a recording chamber, and a recording pipette is used to establish electrical continuity with a retinal ganglion cell. Light pulses are applied to generate excitatory post-synaptic potentials in the ganglion cells, which are recorded by the pipette.

小鼠视网膜切片神经元中的光诱发突触后反应

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Experimental ...

七鳃鳗网状脊髓轴突急性分解到从单个功能突触前终端剥离面膜启用记录

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