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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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，其中在电子显微水平已显示出呈现简单的单活性区域21，可以通过贴标签回收囊泡集群被识别带FM 1-43和对象记录图4c和图4d中 。 CA <s达&gt;离子电流可以被记录在个人的突触前末梢图4F剥离面的膜。钙从单个突触前末梢的剥离面的膜的记录2 +电流直接是显著，因为这是第一个这样的记录在中央突触。制剂的生理相关性的事实，轴突保留功能解离后，允许从具有相似的特征，在网状脊髓轴突在完整脊髓端子突触前末梢记录强调。此外，突触前的 Ca 2 +瞬变的网状脊髓轴突已经表征了在完整的八目鳗脊髓通过钙成像13提供一个参考，以验证从急性分离轴突得到的结果。 关键要获得孤立的网状脊髓轴突与功能性突触前末梢在于一系列评论家人相继步骤。组织阻碍脊髓图1a的dorso -内侧区域主要位于巨轴突急性解离已被首先以振动组织切片图2a移除。这使得它更容易使用尽可能少的力机械解离的脊髓。背柱的切片需要，因为任何残留的背脑膜野蛮会妨碍刀片的切削动作完全去除背侧脑膜野蛮的。我们发现，留下对脊髓腹侧脑膜野蛮在切片时有助于保持脊髓的形状和有助于更好地切片。在切片过程中保持张力在脊髓防止刀片可损伤轴突下的组织的任何横向运动或冲击。长1厘米脊髓件提供了良好的长度为切片的组织可被拉伸并牵制维持张力瓦特往往微不足道的切片。监视另一个关键因素是片的深度;太浅片可防止脊髓同时太深片损害网状脊髓轴突的良好分离。一个好的切片深度是其中的背柱除去明显留下腹侧网状脊髓柱损坏。切片的深度具有在给定的有变化的动物之间脊髓厚度的切片工艺来衡量。背柱的去除是最好进行脊髓片图2b的中间部分，因为它允许对未切割的头侧和尾部端，作为处理该离解过程图2e中把握组织。 除去背柱的，处理所述切片脊髓片用胶原酶的混合物（胶原酶IA型溶组织梭菌）和蛋白酶（蛋白酶类型XIV从灰色链霉菌）的酶（1米后克/毫升林格氏液）帮助消化的结缔组织，便于温和解离。 30-45分钟的消化在室温下是理想的解离。七鳃鳗脊髓完全酶解之前已经进行了用胶原酶（2毫克/毫升，30分钟）和蛋白酶的顺序处理（2毫克/毫升，45分钟）以分离脊髓神经元17。序贯疗法与蛋白酶和胶原酶的鸡尾酒疗法中解离网状脊髓轴突没有取得更好的成绩。我们也尝试用不同浓度的分解酶。酶浓度高于2毫克/毫升或消化时间超过45分钟延长导致脊髓失去一致性和差离解。较高的酶浓度和延长治疗周期可能在脊髓完全离解帮助时，需要更小的神经元的收率。网状脊髓轴突与此相反，有一个扩展的STRUcture和治疗超过45分钟是适得其反。保留脊髓有些紧张援建更好的解离。 下一个重要的步骤是切割酶处理的脊髓片的横向束在切片区域的中点到最终的解离力隔离开来的区域包围网状脊髓轴突。此次裁员应该从腹角灰质以网状脊髓轴突的中央腹侧，内侧柱的外侧边缘延伸使他们完好无损图2d。切割横向大片提供了一个焦点，在这迫使脊髓分离将发生在机械分离，便于平滑的分离组织。在脊髓持续张力在切割过程中是必需的，并且可以通过拉伸和钉扎脊髓片的尾部和喙端部来实现。这防止了手术刀刀片下组织变形和轴突造成的危害。 在离解的最终步骤是施加机械张力并轻轻拉伸沿rostro-尾轴图2e用钳子夹住的组织的组织。进行过聚赖氨酸包被的盖玻片此步骤，以允许分离的轴突用于随后记录的密合性。为了实现对长扩展网状脊髓轴突更大的附着力，我们已经使用了一种高分子量（&gt; 300,000）的聚-D-赖氨酸氢溴酸盐，它提供了更大数量的连接点，以涂覆的盖玻片和陪替氏培养皿的插图。聚赖氨酸包被的表面上提供足够的粘合力为脊髓结束，急性分离网状脊髓轴突。特氟隆涂层镊子需要的抓地力脊髓，因为它牢牢地粘到正规的不锈钢镊子阻碍解离。组织必须慢慢拉长，轻轻拉出来轴突的SP伊纳勒线。获得轴突定居下来的最好的办法是保持沿在任何时候都在离解过程中的玻璃盖玻片通过向下保持压力到喙和尾部脊髓与钳子结束脊髓结束。 轴突是最好划分为局部分离的自轴突的某些部分的内部保持在脊髓末端取决于离解的程度最小一端。隔离可以通过机械分离的脊髓的两端直到轴突从脊髓的一端完全分开进行。在这种情况下，轴突的一个部分保持在脊髓末端从其中出现，而其余部分被分离出脊髓内部的内侧。轴突的末端开口端视轴突的卫生膜重新密封工艺密封起来。隔离，也可以进行这样的轴突从脊髓，B浮现出UT斯达康尾和脊髓由分离的网状脊髓轴突装置保持连接的喙端部图2E。在这一点上，轻轻地释放压力允许在张力下的轴突放松和环出高产轴突缺乏突触后凸起的隔离区。无论是解离技术产生功能性轴突。在生理温度下的八目鳗是10℃，因此，制剂被允许恢复在10℃下1小时，以允许轴突从急性分离回收。用于电生理记录，解离上进行插入的自定义腔室图聚赖氨酸包被的盖玻片3。七鳃鳗脊髓已被一般由灌注林格氏溶液，预冷却至10℃，通过穿过保持在10±2℃下热电冷却装置。极低的背景噪声需要解决单一通道的Ca <sup&gt;离子电流。灌流产生的伪影，因此备用的冷却系统必须被设计以维持解离制备的温度。因此，录音，进行了含林格氏液静止浴。自定义录音室是专为单声道录音。该腔室有一个中央浴室，其中脊髓解离于林格氏溶液，得到分离的轴突。的冷却套管被设计围绕中心浴室，以保持制剂的温度。浴的温度是由通过通入氮气的压力下，绕过任何机电泵送条件（协议第3.15-3.16）灌流防冻溶液，通过一个热电式冷却装置预冷通过，通过外部冷却夹套维持。 高K +（30毫KCl）中使用去极化（4.1节），使调频染料掺入到轴突在突触外切内吞囊泡。它很难留住插入微电极的孤立的轴突的延长周期，丝毫漂移在轴突位置使从轴突退去的电极。这使人们难以刺激轴突用微电极长时间了。大轴突直径（20-50微米）使得很难使用钨刺激电极刺激。因此，高K +，使用去极化的轴突。在网状脊髓轴突的突触前末梢的直径为1-2微米。 的 Ca 2 +电流被定位于在突触前末梢的剥离面的膜，并且因此至关重要的准确定位的突触前末梢。因此，为了实现这些记录的一个关键要求是能够操纵以μm为单位的膜片电极位置的移动。 钙离子电流，在突触前释放的脸膜已经正在示威编在肾盏突触是非常小的幅度，小于0.5 pA的。钙的单声道录音离子电流，因此需要非常低的背景噪声水平。低的热噪声是用Axopatch 200B放大器和冷探头来实现的。此外，噪声污染的来源需要进行系统地识别和消除。补丁移液器是捏造，从铝硅酸盐玻璃，确保超低噪音，因为玻璃具有极低的杂质和高电阻率。被创建的移液管，使得移液管尖直径比的突触前末梢，从而完全包围的突触前末梢的直径大，其尺寸可清楚地由FM 1-43标记观察。这确保了从整个剥离面的膜的记录。电容性噪声降低了涂敷PDMS尽可能靠近尖端尽可能的吸移管。在千兆欧姆密封在突触前末梢的膜是不稳定的长每的IOD和录音通常会持续最多2分钟。这使得该记录极难获得，因为成功率低就必须有大量的尝试，以获得成功的唱片。记录解决方案，必须设计成使得所有其他已知的电流，在突触前末梢膜如电压-门控Na +和K +通道和钙激活钾通道被阻塞，使得记录的 Ca 2 +电流。 有一定的局限性，以使用聚赖氨酸的粘合剂表面与此制剂。一个是不能移动至含有制剂，因为任何振动破碎的制备腔室的物理位置。粘接面的另一个限制在免疫组化实验中发现，由于喙和尾部脊髓末端件失去附着力的时候在4％多聚甲醛培养。我们尝试另一种胶粘剂COA吊环，如细胞德和遇到类似的问题。为了解决这些问题，我们用液体缝合胶水贴上脊髓手柄。关于使用的胶水缝合具体由陈羽和22以前的朱庇特的文章进行了讨论。胶套以较慢的速度解决方案，而二价离子，我们采取了财产的优势，开展林格氏液的离解无钙离子和镁离子 。这在分解过程中为我们提供了更多的时间进行操作的组织。我们使用补丁移液器和吸口通过连接到补丁吸管管之前胶水涂到上解离表面的特定目标区域进行解离（协议第5.2节）采取了类似的方法，陈费瑟斯22。的离解是如前面所述，唯一的区别是，脊柱下进行在轻轻拖动手柄端在先前放置胶（协议第5.3节）的解离过程ORD两端分别贴上。一旦完全解离，二价离子游离林格氏液更换为含Ca 2 +和Mg林格氏溶液2 +离子通过灌注和制剂被允许之前恢复，在10℃下进行20-30分钟的热电冷却板在进行免疫组化的后续步骤。 该技术的关键含义是钙要求神经递质释放，这不尽管几十年的研究得到彻底解决了精确的理解。在突触前的一过性升高的[Ca 2 +]已被确立为是至关重要的触发释放所有的突触。不过，共识仍然缺乏对钙离子通道，有助于对钙域门relea数SE。 钙电流和电容测量个别突触前末梢释放的脸膜的同时测量将产生一个明确的答案。此外，它可以使突触前钙离子进入和囊泡融合之间的时序关系阐发，使突触延迟的精确测量。无限制无障碍个别突触前末梢释放的脸膜提供应用一系列不同的实验方法来研究神经递质释放过程中的各种组件的能力。这种方法的例子包括使用量子点研究突触囊泡融合单囊泡成像。囊泡融合事件也可以通过测量所依据囊泡融合事件的膜电容的变化可以直接特征在于，在突触前末梢。

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 border="0" cellpadding="0" cellspacing="0" width="713">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:211px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">0.2 &#181;m syringe filter</td>
			<td style="width:211px;">EMD Millipore</td>
			<td style="width:119px;">SLGV004SL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">22x60 mm coverslips</td>
			<td style="width:211px;">Fisherbrand</td>
			<td style="width:119px;">12545J</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Advasep-7</td>
			<td style="width:211px;">Cydex Pharmaceuticals</td>
			<td style="width:119px;">ADV7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Alexa Fluor 488 Phalloidin</td>
			<td style="width:211px;">Invitrogen/Life Technologies</td>
			<td style="width:119px;">A12379</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Alexa-633 conjugated goat anti-rabbit secondary antibody</td>
			<td style="width:211px;">Invitrogen/Life Technologies</td>
			<td style="width:119px;">A21070</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Antifreeze</td>
			<td style="width:211px;">Prestone</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Boric acid</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">B7660</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Bright field light source</td>
			<td style="width:211px;">Dolan-Jenner</td>
			<td style="width:119px;">Fiberlite 180</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Calcium chloride</td>
			<td style="width:211px;">Sigma Aldrich</td>
			<td style="width:119px;">C4901</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Collagenase Type IA from Cloristridium histolyticum</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">C9891</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Cover slip</td>
			<td style="width:211px;">Fisher-Scientific</td>
			<td style="width:119px;">12-545-J</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dextrose</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9559</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Digital CCD Camera</td>
			<td style="width:211px;">Hamamatsu</td>
			<td style="width:119px;">C8484-03G01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dissection fine forceps</td>
			<td style="width:211px;">Fine Science Tools</td>
			<td style="width:119px;">91150-20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dissection forceps</td>
			<td style="width:211px;">Fine Science Tools</td>
			<td style="width:119px;">11251-20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dissection microscope</td>
			<td style="width:211px;">Leica Biosystems</td>
			<td style="width:119px;">Leica MZ 12</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dissection scissors</td>
			<td style="width:211px;">Fine Science Tools</td>
			<td style="width:119px;">15025-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dissection scissors fine</td>
			<td style="width:211px;">Fine Science Tools</td>
			<td style="width:119px;">91500-09</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dissection scissors ultra fine</td>
			<td style="width:211px;">Fine Science Tools</td>
			<td style="width:119px;">15000-08</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">FM 1-43</td>
			<td style="width:211px;">Invitrogen/Life Technologies</td>
			<td style="width:119px;">T3163</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Glycine</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">G7126</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">HEPES</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">H7523</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">High vacuum grease</td>
			<td style="width:211px;">Dow-Corning</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Hydrochloric acid</td>
			<td style="width:211px;">Fisherbrand</td>
			<td style="width:119px;">SA-56-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Immersion oil</td>
			<td style="width:211px;">Fisher-Scientific</td>
			<td style="width:119px;">M2000</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Industrial grade Nitrogen gas tank</td>
			<td style="width:211px;">Praxair</td>
			<td style="width:119px;">UN1066</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Insect pins</td>
			<td style="width:211px;">Fine Science Tools</td>
			<td style="width:119px;">26002-10</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Liquid suture glue</td>
			<td>Braun Veterinary Cair Division</td>
			<td style="width:119px;">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 height="20">
			<td height="20" style="height:20px;width:216px;">Magnesium chloride</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">M2670</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Methanol</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">154903</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Non-fat dry milk</td>
			<td style="width:211px;">Cell Signaling Technology</td>
			<td style="width:119px;">9999S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">P-87 Micropipette puller</td>
			<td style="width:211px;">Sutter Instruments</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Paraformaldehyde</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6148</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Perfusion pump</td>
			<td style="width:211px;">Cole-Palmer</td>
			<td style="width:119px;">Masterflex C/L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Petri dish 100x15 mm</td>
			<td style="width:211px;">Fisher-Scientific</td>
			<td align="right" style="width:119px;">875712</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Petri dish 35x10 mm</td>
			<td style="width:211px;">Fisher-scientific</td>
			<td align="right" style="width:119px;">875712</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Poly-D-lysine hydrobromide</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">P1024</td>
			<td>MW &#62; 300000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Potassium chloride</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9333</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Potassium phosphate monobasic</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">P5379</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Primary antibodiesR-type calcium channel</td>
			<td style="width:211px;">Alomone Labs</td>
			<td style="width:119px;">ACC-006</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Protease Type XIV from Streptomyces griseus</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">P5147</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Scalpel blades</td>
			<td style="width:211px;">World Precision Instruments&#160;</td>
			<td align="right" style="width:119px;">500240</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Schot Duran Preesure Bottle</td>
			<td style="width:211px;">Fisher-Scientific</td>
			<td style="width:119px;">09-841-006</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Silicone tubing for glue application</td>
			<td style="width:211px;">Cole-Palmer</td>
			<td>07625-26</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Slicing base plate</td>
			<td style="width:211px;">Leica Biosystems</td>
			<td align="right" style="width:119px;">14046327404</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Slicing chamber</td>
			<td style="width:211px;">Leica Biosystems</td>
			<td align="right">1.4046E+10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Sodium chloride</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7653</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Sodium hydroxide</td>
			<td style="width:211px;"></td>
			<td style="width:119px;">S8045</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Sodium phosphate dibasic</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9763</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Sodium tetraborate</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">B3545</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Sylgard 160 Silicone Elastomer Kit</td>
			<td style="width:211px;">Dow Corning&#160;</td>
			<td style="width:119px;">SYLGARD&#174; 160</td>
			<td>To prepare, mix elastomer A and elastomer B 10:1 by weight</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:211px;">Dow Corning</td>
			<td style="width:119px;">SYLGARD&#174; 184&#160;</td>
			<td>To prepare, mix elastomer and curing agent 10:1 by weight</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Teflon coated forceps</td>
			<td style="width:211px;">Fine Science Tools</td>
			<td style="width:119px;">11626-11</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tricaine methanesulphonate</td>
			<td style="width:211px;">Sigma-Aldrich</td>
			<td style="width:119px;">A5040</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Vibratome blades</td>
			<td style="width:211px;">World Precision Instruments&#160;</td>
			<td style="width:119px;">BLADES</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Xenon lamp</td>
			<td style="width:211px;">Nikon</td>
			<td style="width:119px;"></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造和填充的Alexa Fluor 488的酰肼。轴突和无染料渗漏内均匀分布的染料从轴突图4a中观察到。 /ftp_upload/51925/51925fig4highres.jpg"WIDTH ="500"/&gt; 图从一个孤立的网状脊髓轴突代表性的录音。 （一）隔离网状脊髓轴突充满的Alexa Fluor 488酰肼与补丁吸管在整个小区配置。吸取位置显示一个白色箭头。（b）本人 。代表动作电位发射在一个孤立的网状脊髓轴突。二。代表动作电位发射的网状脊髓轴突的完整脊髓。黑色箭头表示刺激的时间点（C）和（d）分离的网状脊髓轴突表示具有FM 1-43突触前末梢的点状标记与单个突触前末梢的与膜片电极对细胞贴附膜片记录定位的两个图像。绿色箭头标记单个突触前的终端，而白色箭头标志的补丁吸管。（五）死隔离网状脊髓轴突显示indiscrFM 1-43 iminate纳入整个轴突。 4023。示出的 Ca 2 +电流的细胞贴附记录有代表性的例子中（10mM的[Ca 2 +] 外部在补丁吸管记录液）从突触前释放面的膜表现出多种的 Ca 2 +通道的开放。实线标记0表示信道的闭合状态，而虚线表示的高斯峰的通道开口。 急性分离神经轴突保持电性能。静息膜电位（RMP）可以通过刺穿的解离轴突用微电极或补丁在整个小区配置的轴突在电流钳模式下进行测量。类似的风险管理计划都记录在这两种方法中，平均值为-57.94±1.63毫伏（N = 17轴突）。另外，解离的轴突可以刺激射击动作电位图4b，与动作电位是类似的形状和时间进程一烧了网状脊髓轴突在完整的脊髓。 囊泡融合和再造坚持游离轴突。回收囊泡集群可以标记与苯乙烯基染料的FM 1-43在高钾（30 mM的氯化钾）刺激19。当染料的间隙，突触前的终端不同的点状标记可以观察到图4c和图4d，在完整的脊髓13类似网状脊髓轴突的分布。 FM 1-43染色提供了一个明确的区分健康的和不健康的轴突之间不健康的轴突显示整个轴突图4E滥调频染料进入。要明确各个突触前终端的能力，使我们能够针对他们的补丁吸管记录图4c和图4d。利用这种能力，我们从剥离面米记录钙电流直接embrane单突触前末梢图4F。 在急性分离的网状脊髓轴突制剂也具有明显的优势相较于完整的脊髓的单突触前末梢免疫组化。任何背景的自发荧光的缺乏和没有染色突触后结构或神经胶质细胞可用于明确识别抗体标记的鉴定突触前终端（ 图5b，I）。再加上突触前标记如的Alexa-488缀合的鬼笔环肽（ 图5a，ii）和（ 图5b，ⅱ），该标签的突触前肌动蛋白20免疫组织化学标记对突触前末梢可清楚显示的定位（ 图5a，ⅲ）。此方法已被用于结合共聚焦显微镜进行不同VGCC亚型示吨的免疫组织化学鉴定继承人的具体定位到突触前末梢图5a。 <img alt="图5" fo:content-width="6in" src="/files/ftp_upload/51925/51925fig5highres.jpg" width="600" /> 图5免疫染色孤立的网状脊髓轴突。 （一）个别突触前端子R型（CaV2.3）钙通道的免疫组化鉴定。岛多克隆第一抗体被用来在域II和R-型钙通道的第III之间的细胞内环标记的表位（主机 - 兔）。二级抗体是将Alexa Fluor 633的酰肼偶联的山羊抗兔IgG。二。确定突触前肌动蛋白的Alexa Fluor 488鬼笔环肽标记突触前终端。 III。 R型抗体标记（红色）和Alexa-488鬼笔环肽（绿色）的合并叠加显示的共存。（b）本人 。抗R-型钙离子通道抗体与预培养对照抗原不产生钙离子通道的任何特定的标签。二。对于相同的轴突表示离散的点状标记的突触前末梢的的Alexa Fluor 488鬼笔环肽标记。

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

Nanyang Technological University

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

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an extensively studied model system for understanding the behavior of neural stem cells and the regulation of adult neurogenesis. Traditionally, these studies have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region. Compared to sections, wholemounts preserve the complete cytoarchitecture and cellular relationships within the SVZ. This approach has recently revealed that the adult neural stem cells, or type B1 cells, are part of a mixed neuroepithelium with differentiated ependymal cells lining the lateral ventricles. In addition, this approach has been used to study the planar polarization of ependymal cells and the cerebrospinal fluid flow they generate in the ventricle. With recent evidence that adult neural stem cells are a heterogeneous population that is regionally specified, the wholemount approach will likely be an essential tool for understanding the organization and parcellation of this stem cell niche.

The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.

脑室下区恩的脸：Wholemount染色和室管膜流量

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

科研

神经科学

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 ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

电压敏感染料记录单个神经元在大脑切片的轴突，树突和树突棘

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

测量脊髓突触前抑制小鼠背根电位记录<em&gt;在体内</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

尼古丁诱导的钙信号和神经递质的释放了沿着腹侧海马轴突实时成像

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

正常小鼠和亨廷顿小鼠急性脑切片中谷氨酸释放和服用的单突触指标

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our understanding of the molecular mechanisms underlying axon degeneration remains limited. Injury-induced axon degeneration serves as a simple model to study how severed axons execute their own disassembly (axon death). Over recent years, an evolutionarily conserved axon death signaling cascade has been identified from flies to mammals, which is required for the separated axon to degenerate after injury. Conversely, attenuated axon death signaling results in morphological and functional preservation of severed axons and their synapses. Here, we present three simple and recently developed protocols that allow for the observation of axonal morphology, or axonal and synaptic function of severed axons that have been cut-off from the neuronal cell body, in the fruit fly Drosophila. Morphology can be observed in the wing, where a partial injury results in axon death side-by-side of uninjured control axons within the same nerve bundle. Alternatively, axonal morphology can also be observed in the brain, where the whole nerve bundle undergoes axon death triggered by antennal ablation. Functional preservation of severed axons and their synapses can be assessed by a simple optogenetic approach coupled with a post-synaptic grooming behavior. We present examples using a highwire loss-of-function mutation and by over-expressing dnmnat, both capable of delaying axon death for weeks to months. Importantly, these protocols can be used beyond injury; they facilitate the characterization of neuronal maintenance factors, axonal transport, and axonal mitochondria.

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Here, we provide protocols to perform three simple injury-induced axon degeneration (axon death) assays in Drosophila melanogaster to evaluate the morphological and functional preservation of severed axons and their synapses.

在果蝇黑色无gaster的Axon死亡期间，对Axon及其突触的形态和功能评估

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our ...

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a premise for determining the role of neuron types within a neural circuit, and their significance in information encoding relative to learning. Likewise, the method can be used to test the physiological significance of two or more connected brain regions in awake and anesthetized animals. The current study demonstrates how VTA glutamate neurons modulate the firing rate of putative pyramidal neurons in the CA1 (hippocampus) of anesthetized mice. This protocol employs adeno-associated virus (AAV)-dependent labeling of VTA glutamate neurons for the tracing of VTA presynaptic glutamate terminals in the layers of the hippocampus. Expression of light-controlled opsin (channelrhodopsin; hChR2) and fluorescence protein (eYFP) harbored by the AAV vector permitted anterograde tracing of VTA glutamate terminals, and photostimulation of VTA glutamate neuron cell bodies (in the VTA). High-impedance acute silicon electrodes were positioned in the CA1 to detect multi-unit and single-unit responses to VTA photostimulation in vivo. The results of this study demonstrate the&#160;layer-dependent distribution of presynaptic VTA glutamate terminals in the hippocampus (CA1, CA3, and DG). Also, the photostimulation of VTA glutamate neurons increased the firing and burst rate of putative CA1 pyramidal units in vivo.

The current protocol demonstrates a simple method for tracing of ventral tegmental area (VTA) glutamate projections to the hippocampus. Photostimulation of VTA glutamate neurons was combined with CA1 recording to demonstrate how VTA glutamate terminals modulate CA1 putative pyramidal firing rate in vivo.

在河马区文特尔区谷氨酸终端的 Vivo 解剖和功能跟踪组合

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...