我们感谢汉斯·莱纳圩田，马丁FUHRMANN和沃克杰克逊仔细阅读手稿。北莱茵 - 威斯特伐利亚州（SR），BMBF的Projekträger的DLR美中德合作在计算神经科学（CRCNS SR），在神经退行性疾病中的卓越中心（COEN研究MIWF的，由财政部提供的资金; SR），波恩大学校内资助计划（BONFOR SR）。

1。系统要求<ol><li>显微镜系统:枝晶的适合的可视化是至关重要的。如果可用，使用双光子激光扫描显微镜系统。在我们的实验中，我们使用了TRIM范围II LaVision生物技术，比勒费尔德，德国以及一个Ultima IV系统，草原科技，米德尔顿，威斯康星配备钛:蓝宝石超快脉冲激光，相干（变色龙超二）和高NA物镜（60X，0.9的NA，奥​​林巴斯）的可视化充满了补丁吸管通过用荧光染料的树突。虽然被认为是不太严重的，使用双光子扫描光损伤，降低激光功率（低于8 mW在组织）和停留时间（小于1微秒）或尽可能地。 </li><li>第二种可能性是与同步采集，以减少曝光时间尽可能地触发一个宽视场荧光光源（LED或荧光灯）。与集成光源（TILLPho的，我们已经使用了单色补药，德国Grafelfing）的Zeiss Axioskop 2 FS直立显微镜配备与DODT对比度的红外照明（TILLPhotonics，德国Grafelfing）的。在我们的实验中的曝光时间范围通常为10毫秒到最大。 30毫秒。 </li><li>使用快速的微离子导入放大器， 例如双通道微离子导入系统MVCS-C-02（NPI电子，德国Tamm）快电容补偿。细尖离子导入微电极电阻25 - 100MΩ（关键取决于针尖大小和吸管状），如果电容补偿离子电渗放大器的优化调整，才能实现快速上升时间。这种快速的补偿是必要的亚毫秒范围中的离子电渗疗法的移液管的一个简短的，起效快，从而弹出的发射机以高空间分辨率在光斑尺寸小于1μm13施加电流脉冲。电离子透入放大器如此可从其他几家公司，这是我们没有在我们的实验室测试。这些设备是据我们所知，没有配备电容补偿电路。 </li></ol> 2。准备解决方案<ol><li>准备人工脑脊液（ACSF）和内部的解决方案，因为它是所需的实验设计。唯一的内部的解决方案，该解决方案是必需的，除了是红色或绿色的荧光染料（ 例如 50〜200微米的Alexa Fluor 488或594的酰肼，Invitrogen公司），根据光学设备。下面以一个例子学联蔗糖可用于夹层，以mM计:60的NaCl，100蔗糖，2.5氯化钾，1.25的NaH 2 PO 4，26的NaHCO 3，1的 CaCl 2的MgCl 2，20葡萄糖，5;和正常ACSF以mM膜片钳实验:125 3氯化钠，氯化钾，1.25的NaH 2 PO 4，26，2.6的NaHCO 3的 CaCl 2，1.3的MgCl 2，15葡萄糖溶液。 </li><li>Carbogenize（95％，5％的CO 2 O 2）外所有的解决方案不断。 </li><li>准备内部的解决方案，例如，以mM计:140 K-葡萄糖酸，氯化钾，5 HEPES酸，0.5的MgCl 2，5磷酸肌酸，0.16 EGTA; 50 - 200μM的Alexa 488。 </li><li>对于谷氨酸微离子电渗疗法用150mM的谷氨酸，制备溶液，用NaOH将pH调节至7.0。加50元-200微米的Alexa Fluor 488或594肼（Invitrogen公司）的可视化。 </li><li>对于γ-氨基丁酸的离子电渗疗法制备1M的γ-氨基丁酸的溶液和15用HCl将pH调节至5。收费是，在此pH值γ - 氨基丁酸，才可以采用离子电渗疗法。请注意，在溶液中的低pH值可能影响GABA传输本身16,17排出到细胞外空间。 保护GABA的光解决方案和经常准备新鲜GABA原液的，因为旧的解决方案可能会失去其有效性。 </li><li>如果它是很难看到GABA能活动，一喜GH Cl -的驱动力内部的解决方案，例如，离开了氯化钾，可能有助于GABA能以可视化的事件，看是否GABA离子导入工作;然而，调查突触融合的生理驱动力可能被推荐。小GABA能事件对于一般的检测图8C中所示，一个协议可以提供帮助。 </li></ol> 3。拉和测试离子导入移液器<ol><li>在一般情况下，右拉移液器也许是最关键的一步，实现可控的神经递质离子导入。使用离子导入细胞培养时，它是可以拉很细的电极类似尖锐的微电极10。然而，在急性切片膜片钳实验中，这些非常薄的移液管的弯曲的片表面上，当它们被降低到组织的角度，使得不可能达到更深的枝晶。 </li><li>因此，一个非常小的提示，所以拉吸管否Neurotransmitter泄漏出来，但仍然具有足够的刚性，以渗透到组织（ 图2）的前端。例如，使用150 GB F的8P类移液器（科学产品，霍夫海姆，德国）和水平拉马（例如一个DMZ通用型拔轮器，塞特兹仪器公司，Martinsried，德国或P-97式拔轮器，萨特仪器公司诺瓦托，CA）与几个拉一个陡峭的角度（ 图2和表2）的步骤来实现一个小口，但也只有很短的攻丝。 </li><li>另外，也可以使用石英玻璃移液管式离子电渗移液管10。这些都应该有更好的机械性能和更可靠，但是需要特殊的激光拉制机。但它也可以与正常的玻璃移液管，这通常是用于拉补丁移液器达到良好的效果。 </li><li>测试的移液管的性能和无组织的腔室中的电阻，在使用它们之前的第一时间，因为谷氨酸泄漏可能损害组织。 </li><li>正确设置离子导入放大器。然后填写吸管的神经递质和染料溶液，并把它放到洗澡（学联）。 </li><li>补偿电容（ 图3）。通常非常尖锐的移液器不是生硬的将有一个更高的电容。 </li><li>检查移液管电阻:，微离子电渗疗法的放大器，用在这里有一个内置的吸液管电阻测量功能。它唤起了简短的矩形脉冲测试，可以监测一个标准的示波器或采集软件的计算机连接到A / D板。的形状和大小的前端的前端之间的电阻25 - 90MΩ。 </li><li>专注于尖端与60X或40X的水浸泡的目的和开关荧光成像，如果可能的话，如果荧光染料泄漏出枪头放大，适用于一个小的正（谷氨酸的情况下玉手）或负（γ-氨基丁酸的情况下， 图4）保持电流（&lt;0.02μA）。如果不取消的泄漏，更改吸管。 </li><li>申请步骤强大的电流，或使用手动触发和监控尖端的荧光图像中看到，如果该解决方案可以被弹射出的吸管。如上文所述，根据电荷的分子应该要喷射的电流脉冲的极性。要弹出谷氨酸，这是一个负电流和为GABA正电流（ 图4）。 </li><li>气泡中的移液管，该块的染料喷射器和发射器，可以通过施加高喷射电流几次清零。 </li><li>两者合计，如果没有可见的泄漏测试喷射是成功的，补偿电容，并且开始实验。 </li><li>注意:由于电容补偿是通过反馈电路，该电路可过冲或振荡，如果它是overcompensated。轻轻使用​​拨号设置电容补偿。 </li><li>根据对牵拉稳定性，有时是需要不时调整牵拉设置，由于灯丝可能已经改变的属性。但是，如果一次良好的吸液管的设计，可以使用数天。因此，实验完成后存储在密闭容器中的离子电渗疗法的吸管而不损坏小费。 </li><li>在接下来的实验中填写的吸管与神经递质的解决方案，适用于一些强大的弹出脉冲清除小费，然后检查的属性（ 如电阻）的变化明显。然后，补偿电容，并再次使用移液器。不要使用一个单一的吸管有不同的神经递质的解决方案。 </li></ol> 4。准备的脑切片<ol><li>如果微离子电渗疗法是用于第一次肯定准备切片建立可靠工作牵拉程序后。 </li> &lt;根据动物保健机构或地方当局的指引的LI&gt;执行麻醉和斩首程序。 </li><li>搬迁后的大脑，它转移到冰冷学联蔗糖 （见2.1协议）。 </li><li>感兴趣区域的剪切成合适的厚度（例如300微米）的片。 </li><li> ACSF 蔗糖中的切片，在35℃下孵育30分钟。随后，将它们传输到水下保持腔室含有在室温下正常ACSF。 </li><li>在整个筹备和实验carbogenize学联周围的片，用95％5％O 2，CO 2。 </li></ol> 5。建立了全细胞记录<ol><li>离子透入吸管（S）已接近前片表面修补细胞定位，以避免长期持久的定位，建立全细胞模式后。 </li><li>拉出一个低电阻的补丁吸管（3 - 5MΩ），填充的d你们内部解决方案和应用正压移液器（30 - 60毫巴）。 </li><li>进入浴缸和方法下的单元格视觉指导（的红外DODT对比或两个光子梯度对比度的图像）的。 </li><li>监视电压钳模式的测试脉冲（ 例如，-10 mV时，20毫秒）的吸液管电阻。当接触组织纠正偏移电位。 </li><li>接近细胞，轻轻推到它，直到可以清楚地看到"酒窝"枪头。立即释放压力的移液管，适用于40 - 60毫巴的负压，膜电位切换到-65 mV。 </li><li>当电流达到低于100帕的值，释放负压力。 </li><li>建立千兆印章后（电阻&gt; 1GΩ），膜破裂短，吸力强劲的吸管或短暂的过度补偿电容补偿电路。 </li><li>不同的模式（钳位电压或电流）是必需的实验设计，适当的补偿。 </li><li>开始带来离子电渗吸管到其最终位置。如果需要更详细的说明如何成功地执行膜片钳记录，有几个优秀的指引18,19。 </li></ol> 6。将离子导入吸管和生成突触后离子导入势<ol><li>在一般情况下，离子电渗疗法的事件可在规定的地点，这取决于所需的实验，例如，谷氨酸微离子电渗疗法在刺状枝晶，在树突状轴，胞体或轴突起始段为GABA的微离子电渗疗法诱发。 </li><li>接近细胞约1微米的距离，而不触及它。后到达的位置还没有神经递质至关重要的是，泄漏和吸液管电容是否正确补偿。 </li><li>如果接近细胞谷氨酸充满离子电渗pipett的E使检测到膜电位去极化，调整如果可能的话，留住现有的或改变吸管。 </li><li>应用潜在的负电流脉冲，从0开始，并增加当前系统（ 如 0.1 - 0.4毫秒，0.01 - 1μA脉冲）。这有助于找出在该范围内的离子电渗电流唤起的具体的实验中，设定所需的响应率。 </li><li>如果没有响应检测，解除移液器几百微米，并运用强大的弹出电流（&gt; 0.1μA）清洁小费。调整电容补偿，向单元格，然后再试一次。 </li><li>如果仍然没有反应，降低保持电流。与拨号设置要非常小心，因为这个程序可以引起神经递质释放失控。因此，不断地监测记录检测各自的膜电位变化。 </li><li>如果它是很难检测的γ - 氨基丁酸能的事件，也可以是有利于使用n内部的解决方案组成一个高氯产生-驱动力。为了实现这一目标，减少的Cl -浓度液（见协议第2.6节）。这将导致更大的GABA能由于到一个更高的驱动力的静息膜电位的事件。 </li><li>另外，注入长的电流导致膜电位从-100 mV的机会到-48毫伏（ 图8C）的步骤。有了这个协议，Cl -的驱动力增加极化电位，造成一个去极化GABA的响应。 </li><li>另外，也可以使用注入电流的步骤的协议来确定反转电位诱发的事件，这有助于确定事件的GABA能性质。 γ-氨基丁酸的泄漏比谷氨酸泄漏很难被检测到。在这种情况下，输入电阻的恒定监测可以帮助，当γ-氨基丁酸的充满移液管接近。如果输入电阻减小，保持电流可以增加或应使用不同的吸管。 </li><li>作为控制实验谷氨酸离子导入，我们建议的Ca 2 +成像实验中，使用200微米OGB-1并没有EGTA移液器溶液中观察局部钙离子内流，可能是由于泄漏谷氨酸。 </li><li>在一般情况下，实现了稳定的响应，这是非常重要的是要有机械稳定的移液管，随着时间的推移，以避免漂移。漂移可以由温度变化引起的，因此建议改用设备至少一个半小时前的测量，以避免热漂移。请务必使用移液器支架和小费真的是固定的集装式密封好。可以加固定支架本身铁氟龙胶带，此外，可以肯定的是没有探头或机械手，这也是一个潜在来源漂移电缆张力。 </li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+the+mode+of+operation+and+the+dimensions+of+the+junctional+regions+at+synapses+and+motor+end-organs.">The relationship between the mode of operation and the dimensions of the junctional regions at synapses and motor end-organs.</a> Proc. R. Soc. Lond. B. Biol. Sci. 148, 38-56 (1958).</li><li>Kwon, H. B., Sabatini, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+induces+de+novo+growth+of+functional+spines+in+developing+cortex.">Glutamate induces de novo growth of functional spines in developing cortex.</a> Nature. 474, 100-104 (2011).</li><li>Fino, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RuBi-Glutamate:+Two-Photon+and+Visible-Light+Photoactivation+of+Neurons+and+Dendritic+spines.">RuBi-Glutamate: Two-Photon and Visible-Light Photoactivation of Neurons and Dendritic spines.</a> Front Neural Circuits. 3, 2 (2009).</li></ol>

作者宣称，他们有没有竞争经济利益。

在这里，我们将解释如何应用快速微离子导入神经递质调查树突上突触整合。这项技术已被成功地用于研究谷氨酸和GABA能突触传递不同的大脑区域在体外和体内 9,20-22。微离子导入已使用超过60年，但早年它主要是用于本地应用神经递质和药物缓慢或中间的时间跨度23或微量注射物质进入细胞24。 微离子导入研究树突状集成放大器，这是配备一个快速的电容补偿高电阻电极10,11引进以来成为了一个特别有趣的工具。通过这种改进，它成为可能应用的简短的矩形离子电渗电流导致突触后反应，RESE更真实mbled突触活动10,25的时间过程。 有什么技术要求和处理的困难，建立微离子导入时？则需要允许补偿电容的优良的离子电渗移液管的高的离子电渗疗法放大器。正确的调整能力补偿是非常重要的，如果需要具有很高的抗微电极一起高速运转。离子电渗电流仪器提供未补偿杂散电容充电，并因此降低应用程序。正确补偿电容是至关重要的，在视频中详细解释。此外，这是至关重要的光学系统给出了一个很好的荧光图像的枝晶和离子电渗疗法的移液管，而不会引起光损伤的组织。理想情况下，使用双光子系统，降低激光功率和驻留时间，尽可能兼容一个体面的图像质量。如果更传统的光源被使用时，确保尽可能地减少曝光时间，例如，通过使用机械快门或电气触发。可能是最关键的一点是吸液管的设计，这将允许定义和具体的应用程序所使用的神经递质。这一步确实需要一定的时间和精力找到最佳的设置。这里，我们目前为谷氨酸和γ-氨基丁酸的离子电渗疗法的协议，如果其他神经递质受体阻滞剂或调制器的使用，它是必要的，该物质是高度浓缩，更重要的是，它的收费是。例如，由于γ-氨基丁酸具有净电荷为零的pH为0，pH值进行调整（见议定书"第2条），导致净正电荷。 当微离子导入，它将提供一个扩展的工具集来研究神经元突触整合。它允许选择性地刺激突触兴趣一个特定的神经递质。使用选择定义的位置上，突触后电流和电位神经递质，可以追究其传播。此外，有可能同时使用多个离子电渗疗法的电极调查整合多个谷氨酸输入或系统改变了相对的时间或事件的强度。 使用微离子导入时，必须考虑到一些一般性的关键点。微离子导入的个人资料后神经递质的浓度是不同的内源性释放。 murnick及其他10释放的离子电渗疗法的小费（约1毫秒）的速度可能是明显慢于突触释放的囊泡，它发生在一毫秒（0.2毫秒）26级分。另外，从突触间隙扩散可能减缓离子电渗疗应用的神经递质浓度的上升和衰减。此外，喷射神经递质的体积是可能高于生理释放的卷，但是，与高电阻电极，单谷氨酸和GABA能postsynapses的的可能被激活使用微离子电渗疗法10-12。在几微米的范围内的一个空间选择性最近也被实现的神经递质是3,4,27双光子的uncaging的。 虽然相当多的成本密集的，两个光子的uncaging的微离子电渗疗法的几个优点。尤其是，它可以用来释放神经递质，同时在多个突触位点，并且不需要细电极头的精确定位。然而，它也有选择的方法的一个特定的实验时，需要考虑一些重要的缺点。许多笼化合物有不必要的副作用，如神经递质受体的封锁28。例如，许多谷氨酸和GABA笼的已报道与GABA能抑制干扰。此外，需要双光子的uncaging的相对较高的激光功率，可能会导致在光损伤的突触后神经元，特别是当在几分钟内反复施加刺激。此外，站点在刺激的站点的数量是有限的，而微离子电渗疗法，可以自由选择对整个一个神经元的树突树，例如在前端和基树突，模拟层状突触的输入不同的大脑区域。因为它是目前所使用的大多数组双光子的uncaging，将被限制在一个特定的焦平面和视场的，这取决于所使用的物镜（典型的40X或60X的水浸泡）突触网站。它必须考虑到这两种技术总是导致外受体的激活，这可能使结果的解释更加困难。 微离子导入的另一个缺点是，ø只有可以被激活postsynapse，。如果突触前功能和囊泡释放神经递质的作用，需要设置到实验焦点，当地的电气突触刺激可能是一个方法的选择。然而，电突触刺激的缺点相比，微离子电渗疗法的未知位置的激活突触和轴突从不同的神经元群体的共激活，可能属于其他神经递质系统（ 表1）。 摘要微离子导入的最重要的优势是，在我们看来，它是可以使用几种不同的神经递质，并研究其相互作用对如神经车厢的树突9。

在中枢神经系统的神经元突触输入接收各种薄与支树枝状突起。在那里，大多数兴奋性树突状输入介导的谷氨酸突触。这些突触可以在空间上分布的方式被激活，导致突触后的线性整合的兴奋性突触后电位（EPSPs）。如果同时被激活突触上的枝晶的空间接近，这些兴奋性输入可以集成超线性生成树突状尖峰2-5。 此外，一体化的兴奋性输入依赖于树突树的位置上的输入。信号到达远端一簇地区的更减毒比近端投入由于电缆滤波6。在海马，根尖一簇树突遥远的投入产生比那些近端dendri的由不同的大脑区域TES 7。因此，一个令人兴奋的问题是，如何突触输入处理不同的树突状车厢和，如果树突状整合调节神经元放电以不同的方式输入这些分层的影响。 不仅功能特性，枝晶的形态特征，输入的位置和聚类影响树突集成的兴奋性输入，也从GABA能终端的附加 ​​的抑制性输入关键谷氨酸突触8,9疗效判定。突触融合这些不同的方面可以理想地使用神经递质微离子导入树突域空间定义的应用程序不同的神经递质，它允许研究。我们在这里展示如何成功地建立微离子导入谷氨酸和GABA信号在神经元整合调查。 对于这种应用，细尖用于高电阻移液管填充用浓神经递质的解决方案。在这些移液管的位置接近的细胞，其中的神经递质受体位于外膜。树突分支的一个很好的可视化是必需的。使用荧光染料，通过补丁吸管介绍，这是最好的实现。然后，在很短的（&lt;1毫秒）的电流脉冲（在范围为10 - 100 nA的）用于喷射带电的神经递质分子。这些短脉冲和有效电容补偿，突触后电位或电流可诱发高时空精度，表示兴奋性输入的位置是精确已知的。谷氨酸微离子电渗疗法可以激活突触在一个确定的半径，即小于6微米，如下所示（ 图1 9），但它也未能达到单决议突触10-12。 海涅，M.， 等 </e米&gt;显示的空间分辨率的快速微离子电渗疗法，甚至可以进行调整，以发现尺寸小于0.5微米，是小于与两光子的uncaging的谷氨酸13定期取得的光斑尺寸。快速微离子导入，能够容易地使用两个或两个以上的离子电渗移液管，并将其放置在树突树的不同，甚至是遥远的斑点。以这种方式，整合的兴奋性的事件，包括从不同的路径，可以进行调查。另外，也可以在同一时间使用谷氨酸和γ-氨基丁酸的填充离子电渗移液管。以这种方式，在不同的位置，相对于兴奋性输入（的路径中，路径抑制）GABA能抑制效果进行研究。此外，针对特定的神经元域的interneurons抑制的影响，如远端树突，胞体或轴突14，可以使用GABA离子导入。在培养的神经元，快速微离子导入提供了机会，英伟stigate单突触分布的基本方面更详细10,11在神经元突触的沟通。 在这篇文章中，我们详细展示了如何建立使用谷氨酸和GABA离子导入的急性脑切片，允许调查突触融合的兴奋性和抑制性输入的位置输入，输入的优势，以及定时，单独或相互作用的依赖。我们将指出这种技术的优点和局限性以及如何成功地进行故障排除。

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Two-photon laser scanning microscope (TRIM Scope II), and Ultima IV, Prairie Technologies, Middleton, Wisconsin)</td>
			<td>LaVision Biotec, Bielefeld, Germany</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Two-photon laser scanning microscope Ultima IV</td>
			<td>Prairie Technologies, Middleton, Wisconsin, USA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ti:Sapphire ultrafast-pulsed laser</td>
			<td>Chameleon Ultra II, Coherent</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>60X Objective, NA 0.9</td>
			<td>Olympus</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Zeiss Axioskop 2 FS upright microscope</td>
			<td>TILLPhotonics, Gr&auml;felfing, Germany</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Monochromator</td>
			<td>TILLPhotonics, Gr&auml;felfing, Germany</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micro-iontophoresis system MVCS-02</td>
			<td>NPI Electronics, Tamm, Germany</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sutter puller P-97</td>
			<td>Sutter Instrument Company, Novato, CA</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass filaments (150 GB F 8P)</td>
			<td>Science Products, Hofheim, Germany</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 hydrazide</td>
			<td>Molecular Probes life technologies</td>
			<td>A-10436</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594</td>
			<td>Molecular Probes life technologies</td>
			<td>A-10438</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S7653</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma Aldrich</td>
			<td>P9333</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma Aldrich</td>
			<td>S8282</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma Aldrich</td>
			<td>S6297</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S7903</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma Aldrich</td>
			<td>C5080</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma Aldrich</td>
			<td>M2670</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G7528</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>K-Gluconate</td>
			<td>Sigma Aldrich</td>
			<td>G4500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEPES-acid</td>
			<td>Sigma Aldrich</td>
			<td>H4034</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphocreatin</td>
			<td>Sigma Aldrich</td>
			<td>P7936</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E3889</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glutamic acid</td>
			<td>Sigma Aldrich</td>
			<td>G8415</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>GABA</td>
			<td>Sigma Aldrich</td>
			<td>A5835</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Merck</td>
			<td>1.09137.1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Merck</td>
			<td>1.09108.1000</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

fast micro-iontophoresis of glutamate and gaba: a useful tool to investigate synaptic integration

在神经科学的根本利益之一是理解的兴奋性和抑制性输入以及一体化的结构非常复杂，最终导致神经元动作电位输出轴突树突树。特定突触神经元的输出输入不同的空间和时间参数的影响，目前正在调查， 例如随距离衰减的树突状输入，依赖于位置的互动空间隔离输入，兴奋的集成，线性上GABAergig抑制的影响和非线性集成模式，等等。 具有快速微谷氨酸和γ-氨基丁酸的离子电渗疗法，有可能精确地调查谷氨酸激发和GABA能抑制整合的空间和时间。关键的技术要求是一个触发荧光灯，发光二极管（LED），或双光子SCAnning显微镜可视化树突分支，而不会引入显着的光损伤的组织。此外，这是非常重要的是有一个微离子电渗疗法的放大器，它允许快速电容补偿，高电阻移液器。另一个关键的一点是不由自主地释放移液器在实验过程中没有发射。 一旦建立，这种技术将提供可靠和可重复性高的神经递质信号和位置特异性。相比于谷氨酸和GABA的uncaging的，快速离子导入允许使用两个发射机在同一时间，但在很遥远的地方，没有限制的视野。也有局部电刺激轴突相比的优点:微离子导入输入网站的位置肯定是知道的，这是肯定的，只有利益的神经递质释放。然而，必须考虑到与微离子电渗疗法只postsynapse被激活突触前神经递质的释放方面都没有解决。在这篇文章中，我们将演示如何设置微离子导入大脑切片实验。

一个简单的方法来确定空间传播的离子导入是收回离子电渗吸管逐步从枝蔓，同时喷出谷氨酸保持不变。我们发现，一个微离子电渗疗法的刺激的空间范围有一个直径约12微米（ 图1示出的半径）。依赖于深部组织中的可以使用的离子电渗疗法的移液管的刚性。然而，所需的实验片（ 图2），这是用在这里的离子电渗疗法的移液管，没有限制的穿透深度。相反的光学系统和脑切片的深度的降低分辨率是限制因素。对于一个良好的录音质量是至关重要的变送器无泄漏出来的吸管。在谷氨酸的情况下，可以识别泄漏，如果有一个突然的去极化，当枝晶，用移液管尖接触或如果基准突然变成不稳定的（ 图5）。建立一个稳定的记录之后，有可能唤起定义的振幅，树突状尖峰或动作电位的兴奋性突触后整个的树突树（ 图6）的任何位置上使用了这种技术。的性质的EPSP的，他们的传播和求和，应用谷氨酸快速微离子导入，同时在不同的甚至遥远的地方，可以进行调查（ 图7）。 GABA能的事件可以被单独地或附加诱发谷氨酸的第二吸移管的填充离子电渗移液管与一种高度浓缩的GABA溶液（请参阅:第2协议）和积极的排出电流。有一个简单的协议来确认事件的GABA能的性质，使GABA能的事件更容易检测到，如果不使用高驱动力的内部溶液注入负电流（约1秒;的振幅依赖于输入电阻的细胞），导致在hyperpolarising VO的ltage步骤，从在-100毫伏左右，然后增加为5 mV步骤（ 图8）。在较负电位的GABA能的事件更容易检测到，由于较高的驱动力。如果信号反向计算的Cl -溶液的反转电位附近，这是很可能的，它们是GABA能性的（ 图8）。一个额外的γ-氨基丁酸的微离子电渗疗法的移液管，进行调查，例如，GABA能抑制谷氨酸的事件的影响，如树突状钠/钙尖峰，树突状尖峰和抑制性突触后电位（ 图9）的相对时间，通过改变，有可能这两种事件，或它们的幅度相对位置。 （所有的动物实验，按照动物护理的指引进行，并使用波恩大学，德国神经退行性疾病中心和北莱茵 - 威斯特伐利亚州委员会）。 图1。空间范围确定的电泳弹出谷氨酸移液器回缩。 A）最大强度投影图像的CA1区锥体细胞充满了枝晶100μM的Alexa 594和在初始位置（比例尺8微米）的离子电渗疗法的移液管的双光子。插图显示回缩离子电渗吸管和相应的离子电渗EPSP记录在索玛。箭头表示的离子电渗疗法的移液管尖的位置，B）的距离的关系的离子电渗疗法的移液管尖（≤1μm的相差的枝晶中，n = 6个分支）的初始位置相对的EPSP幅度。有了这一点，我们可以估算出系统缩回移液器谷氨酸传播半径。插图显示有代表性的例子痕迹。误差棒代表意味着±经管。 （改编自米勒等人2012年9，从Elsevier许可转载）。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/50701/50701fig2highres.jpg" src="/files/ftp_upload/50701/50701fig2.jpg" /> 图2。离子电渗吸管应该的样子。 A）红外CCD图像相比离子电渗吸管一个小补丁吸管5.5MΩ使用60X的目标B）离子导入规模吸管使用60X的目标。校准:最小= 10微米。 <img alt="图3" fo:content-width="4in" fo:src="/files/ftp_upload/50701/50701fig3highres.jpg" src="/files/ftp_upload/50701/50701fig3.jpg" /> 图3。电容补偿离子电渗吸管使用构建在测试脉冲（10 nA的，10毫秒，NPI电子，塔姆德国）的重要的是要正确地补偿电容监视正确的吸液管电阻，以确保快速和准确的神经递质的应用程序。 <img alt="图4" fo:content-width="6in" fo:src="/files/ftp_upload/50701/50701fig4highres.jpg" src="/files/ftp_upload/50701/50701fig4.jpg" /> 图4。微离子电渗疗法的原理。 A）示意图的CA1锥体神经元全细胞膜片钳配置和充满谷氨酸离子电渗吸。谷氨酸是带负电荷，因此，施加到吸液管的正向电流将保持它从吸移管泄漏出来:保持电流为正（左图）。要退出谷氨酸从吸管，施加一个负的电流（右图）。谷氨酸在这种方式逼出来的吸管，能唤起在突触后细胞的兴奋性事件B）的C示意图A1锥体神经元全细胞膜片钳配置和充满GABA离子电渗吸。 γ-氨基丁酸是带正电的低pH值。因此，一个负电流将保持它从泄漏出来的吸移管（左图）。要退出GABA的应用正向电流（右图）。在这种方式中，γ-氨基丁酸出来的移液管，并能唤起在突触后细胞的抑制事件。 <img alt="图5" fo:content-width="5in" fo:src="/files/ftp_upload/50701/50701fig5highres.jpg" src="/files/ftp_upload/50701/50701fig5.jpg" /> 图5。好的和坏的离子电渗移液器。 B）A）代表性的例子CA1锥体神经元的树突分支产生的离子电渗谷氨酸EPSP产生离子电渗树突状穗代表性的例子枪头持续温和的谷氨酸泄漏的海马CA1区树突分支。 <html><img alt="图6" fo:content-width="6in" fo:src="/files/ftp_upload/50701/50701fig6highres.jpg" src="/files/ftp_upload/50701/50701fig6.jpg" /> 图6。单谷氨酸微离子导入的代表性结果。充满了谷氨酸）一个补丁CA1锥体神经元的示意图和离子电渗吸管B）EPSP诱发谷氨酸离子导入在CA1锥体神经元C）树突状的Na + / Ca 2 +的秒杀诱发的CA1锥体上的近端树突神经元，较低的跟踪显示用电压曲线的斜率，峰值表示树突状尖峰峰值斜率D）增加时施加的电流的离子电渗疗法的EPSP的幅度将增加，直到它穿过动作电位的阈值。 "FO:SRC ="/ files/ftp_upload/50701/50701fig7highres.jpg"SRC ="/ files/ftp_upload/50701/50701fig7.jpg"/&gt; 图7。双谷氨酸微离子电渗疗法的树突树在不同的位置上的有代表性的结果。 A）示意图一个补丁CA1锥体神经元和两个离子电渗移液器充满谷氨酸，这是定位上的近端，远端树突，分别为B）在近端树突的CA1锥体神经元（1）诱发的离子导入EPSP。 C）在其远端（2）枝晶离子导入EPSP诱发。 <img alt="图8" fo:content-width="5in" fo:src="/files/ftp_upload/50701/50701fig8highres.jpg" src="/files/ftp_upload/50701/50701fig8.jpg" /> 图8。代表性的成果为GABA微离子导入。一）示意图绘制一个补丁CA1锥体神经元和离子电渗吸充满GABA。B）在近端树突的CA1锥体神经元。离子导入IPSP诱发）好电流注入系统补丁吸管通过的CA1锥体神经元，变化幅度确定反转电位的诱发事件（-400 pA的电流注射0 PA）。神器表示GABA离子导入的时间点。诱发事件表示反转电位约为-70毫伏（箭头）。 <img alt="图9" fo:content-width="6in" fo:src="/files/ftp_upload/50701/50701fig9highres.jpg" src="/files/ftp_upload/50701/50701fig9.jpg" /> 图9所示。代表性的成果，同时谷氨酸和GABA离子导入调查一体化的抑制和兴奋。电泳诱发A）一个打了补丁的锥体神经元谷氨酸（绿色）和与GABA（橙色）和一个吸管填充示意图B）在随后的扫描中，树突状尖峰独自较低的痕迹显示的dV / dt，峰表明树突状尖峰C，）电泳诱发的IPSP单独D），谷氨酸和GABA能事件。 <table border="1"><tbody><tr><td></td><td> 位置特异性 </td><td> 变送器特异性 </td><td> 毒性/副作用 </td><td> 突触前刺激 </td><td> 长期实验 </td><td> 成本/复杂性 </td></tr><tr><td>微离子电渗疗法</td><td> + + </td><td> + + + </td><td> + + </td><td> - </td><td> + + </td><td> + + </td></tr><tr><td> 2光子的uncaging的</td><td> + + + </td><td> + + + </td><td> + </td><td> - </td><td> + </td><td> + </td></tr><tr><td>突触的刺激</td><td> - </td><td> - </td><td> + + + </td><td> + + + </td><td> + + + </td><td> + ++ </td></tr></tbody></table> 表1中。比较不同的技术。技术的优点和缺点，以刺激神经 ​​元，根据不同的标准（ - =坏，+ =不是最优的，+ =，+ + + =）。 <table border="1"><tbody><tr><td></td><td colspan="2"> 补丁移液器 </td><td colspan="2"> 离子导入移液器 </td></tr><tr><td></td><td> 预拉P（A）单拉 </td><td> 最后拉P（B） </td><td> 预拉P（A）单拉 </td><td> 最后拉P（B） </td></tr><tr><td>热H </td><td> 700 </td><td> 480 </td><td> 510 </td><td> 600 </td></tr><tr><td>空军预拉力F（TH） </td><td> 018 </td><td> 035 </td><td> 018 </td><td> 008 </td></tr><tr><td>距离阈值（TH） </td><td> 017 </td><td> 012 </td><td> 025 </td><td> 015 </td></tr><tr><td>的延迟Heatstop吨（H） </td><td> 050 </td><td> 030 </td><td> 050 </td><td> 030 </td></tr><tr><td>距离Heatstop S（H） </td><td> 030 </td><td> 000 </td><td> 030 </td><td> 000 </td></tr><tr><td>延迟F（F1） </td><td> 000 </td><td> 136 </td><td> 000 </td><td> 050 </td></tr><tr><td>强制拉1 F1 </td><td> 000 </td><td> 065 </td><td> 200 </td><td> 400 </td></tr><tr><td>距离拉2（F2） </td><td> 000 </td><td> 005 </td><td> 000 </td><td> 001 </td></tr><tr><td>强制拉2 F2 </td><td> 000 </td><td> 080 </td><td> 000 </td><td> 095 </td></tr><tr><td>调整（AD） </td><td> 121 </td><td> 000 </td><td> 121 </td><td> 000 </td></tr></tbody></table> 表2中。举例拉马协议。 &lt;/&gt;对于水平拉马（DMZ通用拔轮器，Zeitz的仪器公司，Martinsried，德国），使用8P GB150F的细丝（科学产品，霍夫海姆，德国）两个补丁和离子电渗移液器的强劲。

Watch this Scientific Journal Video about Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration at JoVE.com

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

在这篇文章中，我们介绍快速微离子导入神经递质的突触后信号的高时空精度的技术调查一体化。

谷氨酸和GABA快速微离子导入:一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

fast-micro-iontophoresis-glutamate-gaba-useful-tool-to-investigate

Nanyang Technological University

Research

JoVE Journal

Neuroscience

19.4K 次观看.  University of Bonn. 在这篇文章中，我们介绍快速微离子导入神经递质的突触后信号的高时空精度的技术调查一体化。

视频: Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent1,2. In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue.
Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions3,4. Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling3-8. For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation7,8. However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance.
Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading9-12, which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas7, 8,13, and can be used to study coupling between other retinal neurons, as described here.

An easy and convenient method to determine the extent of gap junction tracer coupling between retinal neurons is described. This technique enables one to investigate the function of the electrical synapses between neurons in the intact retina under different illumination conditions and at different times of the day and night.

剪切载荷:一个有用​​的工具，用于检查视网膜神经细胞之间的缝隙连接示踪剂耦合程度

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic ...

科研

神经科学

MPI CyberMotion Simulator: Implementation of a Novel Motion Simulator to Investigate Multisensory Path Integration in Three Dimensions

Path integration is a process in which self-motion is integrated over time to obtain an estimate of one's current position relative to a starting point 1. Humans can do path integration based exclusively on visual 2-3, auditory 4, or inertial cues 5. However, with multiple cues present, inertial cues - particularly kinaesthetic - seem to dominate 6-7. In the absence of vision, humans tend to overestimate short distances (&lt;5 m) and turning angles (&lt;30&deg;), but underestimate longer ones 5. Movement through physical space therefore does not seem to be accurately represented by the brain.
Extensive work has been done on evaluating path integration in the horizontal plane, but little is known about vertical movement (see 3 for virtual movement from vision alone). One reason for this is that traditional motion simulators have a small range of motion restricted mainly to the horizontal plane. Here we take advantage of a motion simulator 8-9 with a large range of motion to assess whether path integration is similar between horizontal and vertical planes. The relative contributions of inertial and visual cues for path navigation were also assessed.
16 observers sat upright in a seat mounted to the flange of a modified KUKA anthropomorphic robot arm. Sensory information was manipulated by providing visual (optic flow, limited lifetime star field), vestibular-kinaesthetic (passive self motion with eyes closed), or visual and vestibular-kinaesthetic motion cues. Movement trajectories in the horizontal, sagittal and frontal planes consisted of two segment lengths (1st: 0.4 m, 2nd: 1 m; &plusmn;0.24 m/s2 peak acceleration). The angle of the two segments was either 45&deg; or 90&deg;. Observers pointed back to their origin by moving an arrow that was superimposed on an avatar presented on the screen.
Observers were more likely to underestimate angle size for movement in the horizontal plane compared to the vertical planes. In the frontal plane observers were more likely to overestimate angle size while there was no such bias in the sagittal plane. Finally, observers responded slower when answering based on vestibular-kinaesthetic information alone. Human path integration based on vestibular-kinaesthetic information alone thus takes longer than when visual information is present. That pointing is consistent with underestimating and overestimating the angle one has moved through in the horizontal and vertical planes respectively, suggests that the neural representation of self-motion through space is non-symmetrical which may relate to the fact that humans experience movement mostly within the horizontal plane.

An efficient way to gain insight into how humans navigate themselves in three dimensions is described. The method takes advantage of a motion simulator capable of moving observers in ways unattainable by traditional simulators. Results confirm that movement in the horizontal plane is underestimated, while vertical movement is overestimated.

MPI Cyber​​Motion模拟器:一种新型的运动模拟器的执行情况调查三个维度的多感官的路径积分

Path integration is a process in which self-motion is integrated over time to obtain an estimate of one's current position relative to a starting point 1. ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

突触荧光自动定量<em&gt; C。线虫</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

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

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

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

星形细胞转运电流派生谷氨酸间隙的时间进程与反卷积分析

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

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility1-7. To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent &#945;-bungarotoxin with cells expressing constructs containing an &#945;-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the &#945; subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity8,9. Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors2,10. In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP11) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)12. The BBS is 3&#39; of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

Here we demonstrate the use of fluorescent Alexa dye coupled to &#945;-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds &#945;-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.

使用α-银环蛇毒素结合位点标记来研究GABA A受体膜定位和贩运

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In particular, endogenously-generated network activity, which strongly varies as a function of the state of vigilance, significantly modulates neuronal computation. To investigate how different spontaneous cerebral dynamics impact single neurons&#39; integrative properties, we developed a new experimental strategy in the rat consisting in suppressing in vivo all cerebral activity by means of a systemic injection of a high dose of sodium pentobarbital. Cortical activities, continuously monitored by combined electrocorticogram (ECoG) and intracellular recordings are progressively slowed down, leading to a steady isoelectric profile. This extreme brain state, putting the rat into a deep comatose, was carefully monitored by measuring the physiological constants of the animal throughout the experiments. Intracellular recordings allowed us to characterize and compare the integrative properties of the same neuron embedded into physiologically relevant cortical dynamics, such as those encountered in the sleep-wake cycle, and when the brain was fully silent.

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

This procedure performs long-lasting in vivo intracellular recordings from single neurons during physiologically relevant cerebral states and after complete abolition of ongoing electrical activities, resulting in an isoelectric brain state. The physiological constants of the animal are carefully monitored during the transition to the artificial comatose condition.

等电脑国家的诱导研究内源性突触活动对神经元兴奋性影响<em&gt;在体内</em

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

使用基于酶的生物传感器来衡量进补和阶段性谷氨酸在阿尔茨海默氏症小鼠模型

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

使用快速、简单且便宜的 AWESAM 协议培养在体内像小鼠星形胶质细胞

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

光遗传学与人工 microRNAs 相结合对完整神经元回路中基因击倒对突触前功能的影响

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

谷氨酸和GABA快速微离子导入:一个有用的工具，调查突触整合

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此视频中的章节

剪切载荷:一个有用的工具，用于检查视网膜神经细胞之间的缝隙连接示踪剂耦合程度

MPI CyberMotion模拟器:一种新型的运动模拟器的执行情况调查三个维度的多感官的路径积分

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

突触荧光自动定量

星形细胞转运电流派生谷氨酸间隙的时间进程与反卷积分析

使用α-银环蛇毒素结合位点标记来研究GABA A受体膜定位和贩运

等电脑国家的诱导研究内源性突触活动对神经元兴奋性影响

使用基于酶的生物传感器来衡量进补和阶段性谷氨酸在阿尔茨海默氏症小鼠模型

使用快速、简单且便宜的 AWESAM 协议培养在体内像小鼠星形胶质细胞

光遗传学与人工 microRNAs 相结合对完整神经元回路中基因击倒对突触前功能的影响

谷氨酸和GABA快速微离子导入:一个有用​​的工具，调查突触整合

摘要

探索更多视频

此视频中的章节

剪切载荷:一个有用​​的工具，用于检查视网膜神经细胞之间的缝隙连接示踪剂耦合程度

MPI Cyber​​Motion模拟器:一种新型的运动模拟器的执行情况调查三个维度的多感官的路径积分

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

突触荧光自动定量

星形细胞转运电流派生谷氨酸间隙的时间进程与反卷积分析

使用α-银环蛇毒素结合位点标记来研究GABA A受体膜定位和贩运

等电脑国家的诱导研究内源性突触活动对神经元兴奋性影响

使用基于酶的生物传感器来衡量进补和阶段性谷氨酸在阿尔茨海默氏症小鼠模型

使用快速、简单且便宜的 AWESAM 协议培养在体内像小鼠星形胶质细胞

光遗传学与人工 microRNAs 相结合对完整神经元回路中基因击倒对突触前功能的影响

谷氨酸和GABA快速微离子导入:一个有用的工具，调查突触整合

剪切载荷:一个有用的工具，用于检查视网膜神经细胞之间的缝隙连接示踪剂耦合程度

MPI CyberMotion模拟器:一种新型的运动模拟器的执行情况调查三个维度的多感官的路径积分