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., 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

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

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

科研

神经科学

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

利用生物测定引导分馏法鉴定海洋鳗信息素

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

生物化学

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 ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

细胞内记录，感官字段映射，并在水蛭养殖鉴定神经元，医用水蛭</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

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 ...

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation.

星爆无长突细胞的膜片钳在Deafferentated小鼠视网膜的平板安装准备

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

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 ...

Electrophysiological Investigations of Retinogeniculate and Corticogeniculate Synapse Function

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ganglion cells and project it to the visual cortex. In addition, relay neurons receive top-down excitation from the cortex. The two main excitatory inputs to the relay neurons differ in several aspects. Each relay neuron receives input from only a few retinogeniculate synapses, which are large terminals with many release sites. This is reflected by the comparably strong excitation, the relay neurons receive, from retinal ganglion cells. Corticogeniculate synapses, in contrast, are simpler with few release sites and weaker synaptic strength. The two synapses also differ in their synaptic short-term plasticity. Retinogeniculate synapses have a high release probability and consequently display a short-term depression. In contrast, corticogeniculate synapses have a low release probability. Corticogeniculate fibers traverse the reticular thalamic nuclei before entering the lateral geniculate nucleus. The different locations of the reticular thalamic nucleus (rostrally from the lateral geniculate nucleus) and optic tract (ventro-laterally from the lateral geniculate nucleus) allow stimulating corticogeniculate or retinogeniculate synapses separately with extracellular stimulation electrodes. This makes the lateral geniculate nucleus an ideal brain area where two excitatory synapses with very different properties impinging onto the same cell type, can be studied simultaneously. Here, we describe a method to investigate the recording from relay neurons and to perform detailed analysis of the retinogeniculate and corticogeniculate synapse function in acute brain slices. The article contains a step-by-step protocol for the generation of acute brain slices of the lateral geniculate nucleus and steps for recording activity from relay neurons by stimulating the optic tract and corticogeniculate fibers separately.

Here, we present protocols for the preparation of acute brain slices containing the lateral geniculate nucleus and the electrophysiological investigation of retinogeniculate and corticogeniculate synapses function. This protocol provides an efficient way to study synapses with the high-release and low-release probability in the same acute brain slices.

视网膜原酸和皮质化突触功能的电生理学研究

The lateral geniculate nucleus is the first relay station for the visual information. Relay neurons of this thalamic nucleus integrate input from retinal ...

Activity of Posterior Lateral Line Afferent Neurons during Swimming in Zebrafish

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates reafferent cues that animals must disentangle from relevant sensory cues of the surrounding environment. For example, when a fish swims, flow generated from body undulations is detected by the mechanoreceptive neuromasts, comprising hair cells, that compose the lateral line system. The hair cells then transmit fluid motion information from the sensor to the brain via the sensory afferent neurons. Concurrently, corollary discharge of the motor command is relayed to hair cells to prevent sensory overload. Accounting for the inhibitory effect of predictive motor signals during locomotion is, therefore, critical when evaluating the sensitivity of the lateral line system. We have developed an in vivo electrophysiological approach to simultaneously monitor posterior lateral line afferent neuron and ventral motor root activity in zebrafish larvae (4-7 days post fertilization) that can last for several hours. Extracellular recordings of afferent neurons are achieved using the loose patch clamp technique, which can detect activity from single or multiple neurons. Ventral root recordings are performed through the skin with glass electrodes to detect motor neuron activity. Our experimental protocol provides the potential to monitor endogenous or evoked changes in sensory input across motor behaviors in an intact, behaving vertebrate.

We describe a protocol to monitor changes in the afferent neuron activity during motor commands in a model vertebrate hair cell system.

斑马鱼游泳期间后横向线阿弗伦特神经元的活动

Sensory systems gather cues essential for directing behavior, but animals must decipher what information is biologically relevant. Locomotion generates ...

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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利用生物测定引导分馏法鉴定海洋鳗信息素

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

细胞内记录，感官字段映射，并在水蛭养殖鉴定神经元，医用水蛭

完全性脊髓损伤和脑解剖协议后续Wholemount

星爆无长突细胞的膜片钳在Deafferentated小鼠视网膜的平板安装准备

斑马鱼

爪蟾Tectal 神经元全细胞膜片钳记录的制备与协议

视网膜原酸和皮质化突触功能的电生理学研究

斑马鱼游泳期间后横向线阿弗伦特神经元的活动