We would like to thank all members of &#8216;Function of Neuronal Microcircuits&#8217; Group at Institute of Neuroscience and Medicine, INM-2, Research Centre J&#252;lich and the &#8216;Function of Cortical Microcircuits&#8217; Group in the Dept. of Psychiatry, Psychotherapy and Psychosomatics, Medical School, JARA, RWTH Aachen University for fruitful discussions. This work was supported by the DFG research group on Barrel Cortex Function (BaCoFun).

所有的实验过程进行了按照欧盟指令保护动物，德国动物福利法（Tierschutzgesetz）和欧洲实验动物科学协会联合会的指导原则。 1.建立了电与配对记录开始之前，一个电设置具有待建。简述怎么这么的建立组装下面给出: <ol><li>安装一个防振台在其上的显微镜，操纵器和所有其他设备将被放置。 注:振动或任何其它类型的运动需要从突触连接的记录，因为这需要移液器的变化（在搜索电极通过记录电极取代）时，必须尽可能地小。 </li><li>周围放置防振台的法拉第笼，以减少电噪声。连接所有设备法拉第CA内GE与电气接地。 </li><li>安装有一个根据制造商的指导方针的基础上的机动的xy表上的防振台机动焦点轴的显微镜。这允许显微镜可靠地移动到的神经元不属于在同一视场，因为它是在大脑皮层转层连接的情况下。 </li><li>显微镜的相机端口上安装一台摄像机，并连接它到模拟和/或数字的屏幕观察切片的制备和电极的移动。使用照相机，其可以一个低和高功率放大，以获得一个概述和特写图像之间，来切换所述突触耦合的神经元细胞对分别。这也有助于控制块电极的移动。 </li><li>安装含有插入的浴室中的样品台（在内部从一个有机玻璃块钻）的脑切片制备。此标本表不能连接到显微镜， 即，必须将固定在空气台和显微镜应根据它的机动性。 </li><li>摩两个（或更多，如果需要）的高精度微操作，能够在所有三个维度上移动。上的操纵器安装膜片钳前置放大器和它们连接到主放大器。 注:如果需要额外的操纵器可以被安装为例如两个以上的前置放大器，细胞外刺激电极，药物输送系统等。 </li><li>将浴室中的样品台。安装解决方案的入口和出口，并将它们连接到灌注系统。 </li><li>使用蠕动泵以保持该记录室用人工脑脊液的灌注（ACSF; 表1），在4以稳定的流量- 6毫升/分钟为最佳切片生存能力。 注意:不要超过这个流量显著，因为它会导致动荡ASCF和切片，因此运动。 </LI&gt; <li>温度探测器和银/氯化银接地电极到样品表安装持有者。这是必要的，以允许探针和电极样品在室中的浴。 </li><li>对于神经元的切片准备了良好的知名度，在显微镜下用红外线照射。这减少了光的衍射，因此图像模糊。确保该显微镜配备有微分干涉对比光学实现了"3D&#39;状可视化。这将有助于修补神经元，并允许针对神经元深埋在片（60微米或更深）。 </li><li>在实验过程中，保持脑切片在实验室中与在底部的玻璃盖玻片。发布一条"铂竖琴"片上的顶部，以防止片浮动。铂竖琴从U形，扁平铂丝制成与牙线串粘在上面。 </li><li>保持ACSF灌注切片在32的温度下 - 35℃，以确保选择进制控制氧化和pH值。这可以通过加热与安装在靠近ACSF流入浴室中的珀尔帖装置的溶液来完成。 注意:使用超薄盖玻片使得即使显微镜冷凝器具有高数值孔径和工作距离短可以聚焦在试样上。这是必要的切片的最佳照明。 对于图像和一个电设置了配对录音优化大脑切片准备的说明见参考文献34 图4。 </li></ol> 2.脑切片准备<ol><li>轻度麻醉从中脑组织应采取用异氟烷（最终浓度&lt;0.1％）的动物。 注意:其他麻醉剂也可使用。使用麻醉剂应符合相应的动物委员会的建议和规则。始终确保动物不过敏或在压力下加入后，麻醉剂。 </li><li>斩首动物，打开头骨，并使用34,35在参考文献中描述的方法。迅速取出大脑越好。 </li><li>转移到脑冷却的（4℃）的人工脑脊髓充以卡波金的气体用95％O 2和5％的CO 2的混合物的流体。 </li><li>隔离应研究大脑区域。 注:该参数，以获得最佳的保存和树突前和突触后神经元轴突的最小截断应事先根据调查，每个神经元的连接和发展阶段决定的。在一个最佳解剖脑切片，则不需要突触前神经元的轴突平行于片表面。相反，它们应指向与一个小角度的切片。在适用于突触后锥体细胞的顶枝晶;这应该在光学显微镜下进行检查。这是对非本地或叔特别重要ranslaminar突触连接， 即 ，其中前和突触后胞体均超过200微米拆开的树突和轴突截短的概率是可能比用于本地连接基本上更高。 </li><li>脑转移到切片机室中。根据经验发现，不同的细胞外的切片的解决方案依赖于动物的年龄（见表2和3;有用的信息，也可根据&#39;发现<a href="http://www.brainslicemethods.com/" target="_blank">http://www.brainslicemethods.com/</a> &#39;）。 </li><li>修剪大脑直到目标区域是可见的。 </li><li>以获得片具有良好的细胞可见性，切300 - 400微米厚的脑切片，如果动物是成熟（&gt; 3周）。如果动物是未成熟（第1次至2 次产后一周小鼠和大鼠）的切片可以被切割到厚度为〜600 - 800微米，而基本上不损害细胞visibility。 注意:这将提高"不成熟"片的稳定性，减少远距离树突和轴突抵押品可能截断的数量。 </li><li>从切片机腔室转移切片到孵育室填充有通入95％O 2和5％ 的 CO 2的混合切片溶液。保持在培养室中的片至少30分钟至1小时或者在室温或在约30℃，这取决于实验的类型。 </li></ol> 3.配对膜片钳记录和生物胞素填充取决于突触连接的类型三种不同的方法用于查找突触耦合的神经元。如果连接概率很低（因为可以预期最兴奋的连接），步骤如下: <ol><li> 具有低连接神经元的连接 <ol><li>填写贴片移液器与组合物的所列的内部溶液表4对于在全细胞构造的所有录像添加生物胞素在3之间的浓度- 5毫克/毫升到内部（吸管）溶液中，得到良好的染色结果的前和突触后神经元。让生物胞素扩散进入细胞至少15 - 30分钟（取决于神经元的大小）。 注意:不要生物胞素添加到下面提及的"搜索"吸管使用的解决方案。 </li><li>修补在全细胞模式的推定的突触后神经元。使用补丁移液器与修长小腿。这便于吸移管的操作性下的目标，并减少当电极在切片被引入可能发生的运动伪影。 </li><li> 10兆欧电阻和一个小尖端直径 - 然后，用8的"搜索"膜片吸管修补一个潜在的突触前神经元在细胞连接的配置。有了这些吸管建立"宽松"细胞贴附补丁。 FIL升了"搜索"吸管具有内部溶液中的 K +时，为了不期间搜寻在突触前的细胞去极化神经元代替的Na +（ 表5）。 注:"松"密封阻力不在GΩ的范围，但通常为约30 - 300兆欧。 </li><li>握在电流钳模式下的"宽松"封膜电位为约-30至-60毫伏。然后，应用的大电流脉冲（0.2 - 2 NA）引发的潜在的突触前细胞的动作电位。观察这一动作电位作为电压响应小小穗。 </li><li>刺激频率设定为0.1赫兹，以防止一个突触后响应破落。使用较低的刺激频率的情况下的脑组织是非常不成熟或突触连接并不十分可靠。 </li><li>在情况下，"突触后"神经元不向测试"突触前"神经刺激响应罗恩，补丁&#39;宽松&#39;印章模式的新的神经元。只要不更换&#39;搜索&#39;补丁电极为&gt; 30MΩ成立电阻密封。测试多达这种方式30潜在的突触前神经元。 注:为了防止松散膜片钳的搜索过程中损坏的神经元，仅轻轻抽吸，应适用于搜索吸管施加到全细胞膜片吸管比较。 </li><li>如果刺激在&#39;宽松&#39;印章模式导致的EPSP / IPSP有潜伏期&lt;5毫秒，在突触后神经元的突触前神经元中删除"搜索"吸管。 </li><li>更换&#39;搜索&#39;补丁吸管与补丁电极注入含有生物胞素 - ，定期的内部解决方案。确保这个录音补丁吸管有4电阻 - 8MΩ;较大的胞体大小下部电极电阻即可。 </li><li>修补突触前神经元和记录在全细胞，电流钳模式。通过电流注入在突触前神经元诱发动作电位，并记录突触后的反应。存储数据的计算机上购买离线分析。 </li></ol></li><li> 具有高连接神经元的连接 注:对于神经微电路具有高连接率（&gt; 30％），使用不同的程序来找到突触连接。此过程概述如下，并经常被用于具有抑制性突触连接上平均更高的连接比较兴奋性连接36。当连接率低于该值时，它不应该被使用。 <ol><li>直接在全细胞模式修补突触前神经元。然后，修补一个潜在的突触后神经元，也全细胞模式。两个单元应根据目前的钳位控制。引起突触前细胞的动作电位。监测准突触后神经元的postsynaptic潜力。 </li><li>在的情况下，神经元不显示到突触前刺激的响应，用一膜片电极填充有规律的内部溶液修补在全细胞模式的新的神经元。如果发现连接，不改变补丁吸管，但继续进行录制。然后，继续按步骤3.1.9。 </li></ol></li><li> 通过间隙路口的神经元连接 注意:要搜索电气（间隙连接）连接使用下列步骤，它代表用于低概率连接，修改后的版本。 <ol><li>修补在全细胞膜片钳结构的突触后神经元。 </li><li>随后，在打补丁的宽松的密封结构的假定突触前神经元（30个低电阻印章 - 300MΩ）使用的7&#39;搜索&#39;补丁吸管 - 10MΩ的电阻。此吸管应该充满松动密封刺激改性高的Na +内液。 </li><li> Injec吨100 - 300毫秒通过&#39;松&#39;封长超极化电流脉冲;吸移管电位命令应设置为约-60至-70毫伏。注意在"突触后"神经元小超极化反应，间隙连接的连接，如果这种刺激的结果。 </li><li>重配接在全细胞模式的"突触前"神经元，并继续如上所述。 注:为了确保轴突和树突的进程良好的染色，使用下面给出的协议。这个协议在短暂这里描述，但是，对于在我们的实验室中使用组织化学处理的详细协议见参考文献37。 </li></ol></li></ol> 4.组织化学处理<ol><li>与该对突触耦合的神经元传输脑切片成含有4％多聚甲醛溶解在0.1M磷酸盐缓冲液的小瓶中，并固定在切片24小时。电子显微镜，固定用2.5％戊二醛的切片醛和1％多聚甲醛。 </li><li>在第二天，转切片成不含有固定剂的正常的磷酸盐缓冲液中。 8次10 - - 用磷酸盐缓冲液洗6切片15分钟，每一个以除去过量的固定剂。 </li><li>孵育在3％的H 20分钟的切片2 O 2的 0.1M磷酸盐缓冲液以最小化的内源性过氧化物酶反应。观察强泡沫的形成。继续进行反应直到没有进一步的气泡产生。然后，漂洗中为6的0.1M磷酸盐缓冲液的片- 8次（每次10分钟），以除去任何剩余的 H 2 O 2。 </li><li> 免疫组化和显色反应 生物胞素填充神经元的可视化是基于催化剂以二氨基联苯胺一个链霉抗生物素化的辣根过氧化物酶反应;这将产生能产生脆污渍具有高空间分辨率的一个暗沉淀。下列程序被用于显色反应:&lt;OL&gt; <li>准备ABC解决方案按照制造商的协议。加入14.55毫升磷酸盐缓冲液补充有150微升10％的Triton X100（ 表6;仅用于光学显微镜），并保持该溶液为在黑暗中约30分钟。在这个解决方案O / N使用前孵化切片。 </li><li>孵育在振荡器上1小时，在室温和在黑暗中的ABC溶液切片。随后，不断切片O / N在4℃的冰箱。上的第二天早晨，在室温下孵育切片为一小时。 </li><li>在此之后，冲洗切片，至少四次，在磷酸盐缓冲液每10分钟。然后，孵育在振荡器上约30分钟，在黑暗中在2毫升的镍加剧3-3&#39;-二氨基联苯胺溶液（ 表7）。处理氨基联苯胺，只有在通风橱使用时要非常小心;它甚至在非常低的浓度极其致癌性。 </li><li>添加6.5微升3％的H 2 O 2的所述含二氨基联苯胺-解决方案。监视在光学显微镜下对显色反应，直至棕黑色染色的神经元都清晰可见。然后，通过洗涤切片数倍于磷酸盐缓冲液，立即使反应停止。 </li><li>片山与胶粘剂，硅烷涂层Histobond或凝胶化标准载玻片染色的神经元。 </li><li>保持载玻片在保湿室具有至少80％的湿度O / N。在接下来的日子里，让切片风干另外10分钟。之前嵌入，转印滑动随着乙醇浓度的溶液 - 以脱水它们（20 100％）。 注:脱水过程应缓慢否则在树突和轴突过程的扭曲可能发生37。如果一个替代嵌入程序被选择， 例如，一个与甘油基Moviol，脱水不是必需的;然而，这很可能会导致在该切片的不均匀压缩，因而扭曲神经morpholoGY。 </li><li>最后，孵育10分钟，在二甲苯，嵌入切片在光学显微镜疏水包埋介质和放置一个超薄盖玻片放在上面。让载玻片干燥20分钟，在RT。 </li></ol></li></ol> 5.神经重建和突触联系本地化<ol><li>检查载玻片与生物胞素标记的神经元的光显微镜配备一个60X / 100X油浸物镜和与最佳空间分辨率高数值孔径的冷凝器下。确保轴突终扣和树突棘都清晰可见。 </li><li>跟踪使用商业神经追踪系统的神经元或突触耦合神经对（ 参见表具体材料/设备 ），以获得三维神经重建。开展不断受到目视检查手动跟踪检测到小轴突树突或抵押品。从突触神经元加上配对录音手动跟踪依然方法选择，因为例如，一个轴突配置文件无论是前或突触后神经元的归属需要大量的重建经验。 </li><li>如果需要，使轴突终扣和/或树突棘的数量。因为刺甚至脊柱亚型和轴突终扣可以表现出相对于例如，皮质层或区域的特定的分布和密度，这可能有助于表征神经元细胞类型。 </li><li>对于突触神经元加上对，检查突触前神经轴突和树突突触后近距离appositions在可用的最高放大倍率。观察一个轴突布顿和突触后树突棘或轴是在同一焦平面可以被认为是假定的突触接触。纪念这个接触的重建。 </li><li>纠正神经重建收缩在跟踪程序的相应部分的所有三个空间维度。 注:在固定，组织化学处理和脱水显着的机械变形和收缩发生;这会影响轴突和树突长度测量，并严重扭曲了他们的空间安排。收缩的校正因子用于灌封介质是〜1.1为x和y方向和〜2.1为z方向37。 </li><li>对于定量形态分析使用数据分析程序的神经生理学（ 见表具体材料/设备 ）。 </li></ol>

The authors declare no conflict of interest.

从突触耦合兴奋性和/或抑制性神经元成对录音是神经元微电路的研究中非常通用的方法。不仅这种方法允许一个来估计神经类型之间的突触连接，但也允许确定连接和前和突触后神经元的形态的功能特性。此外，激动剂和/或拮抗剂可以很容易地应用到神经元片的准备工作。这允许人们研究神经调的影响突触传递32的属性或一个深入表征使用定义酉突触连接的，例如，突触释放16,17,38,39...

2突触耦合的神经元之间的神经元微电路是在大脑中的大型网络的积木并且突触信息处理的基本单位。一个先决条件，例如神经元微电路的特征是知道的形态和两个前和突触后神经元的伙伴的功能特性，突触连接（S）和它的结构和功能的机制的类型。然而，在突触连接的许多研究中的至少一个在微电路中的神经元的是不是很好的特点。这导致从通常的突触连接研究中使用的相对非特异性的刺激协议。因此，突触前神经元的结构和功能特性要么根本不或仅到一个相当小的范围内确定的（ 即，标记蛋白质的表达等 ）。在组合配对录音与细胞内染色用标志sUCH作为生物胞素，neurobiotin或荧光染料更适合用于研究小神经微电路。这种技术允许一个调查的同时一个形态鉴定突触连接的许多结构和功能参数。 所谓的"单一"突触两个神经元之间的联系进行了研究使用急性切片准备两种皮层和皮层下大脑区域1-10。最初，尖锐的微电极在这些实验中使用;之后，膜片钳记录是采用为了获得突触信号记录具有较低噪声电平以及一个改进的时间分辨率。 一个显著的技术进步是利用红外微分干涉对比（IR-DIC）的光学11-14，微观技术，显著改善神经细胞的知名度和识别的脑片，使之成为可能吨Ø获得录音从视觉上确定的突触连接15-17。在一般情况下，一对录音进行急性切片的筹备工作;只有极少数的出版物可从报告突触连接的神经元在体内 18-20录音。 配对记录的最重要的优点是，一个功能表征可以与同时在光学和电子显微镜水平形态解析被组合（ 例如见。，7,16,21）。组织化学处理后，将突触连接的神经元对的树突和轴突的形态被追踪。随后，有可能量化的形态特征，如长度，空间密度，取向，分支图案等 ，然后这些参数可以为特定的突触连接的一个目标分类提供了基础。此外，与此相反，以用于研究神经元connecti大多数其它技术VITY，配对录音还允许对单一的突触连接突触联系的标识。这可以直接使用光学和电子显微镜16,21-27的组合或使用树突棘的钙成像28,29来完成。但是，与后者的方法只兴奋性但不抑制连接，可以研究，因为它需要通过突触后受体通道的钙内流。 除了 ​​突触传递的在确定的神经元微电路配对的记录进行详细的分析还允许的突触可塑性规则30,31的研究或-与激动剂/拮抗剂组合的应用-突触传递由神经递质如乙酰胆碱32和腺苷调制33。

<table><tbody><tr><td>Amplifier</td><td>HEKA</td><td>EPC 10 USB Triple</td><td>with 2 - 3 preamplifiers</td></tr><tr><td>Microscope</td><td>Olympus</td><td>BX51WI</td><td>with 2 camera ports, a 4X objective, and a 40X water-immersion objective</td></tr><tr><td>Camera</td><td>TILL Photonics</td><td>VX55</td><td>infrared CCD camera</td></tr><tr><td>Workstation</td><td>Luigs &amp;amp; Neumann</td><td>Infrapatch 240</td><td>with a motorized x-y stage and a motorized focus axis for the microscope</td></tr><tr><td>Micromanipulator</td><td>Luigs &amp;amp; Neumann</td><td>SM-5</td><td>x-y-z manipulators for 2 - 3 preamplifiers</td></tr><tr><td>Faraday cage</td><td>Luigs &amp;amp; Neumann</td><td></td><td></td></tr><tr><td>Anti-vibration table</td><td>Newport Spectra-Physics</td><td></td><td></td></tr><tr><td>Patchmaster</td><td>HEKA</td><td></td><td></td></tr><tr><td>Microtome</td><td>Microm International</td><td>HM650V</td><td></td></tr><tr><td>Micropipette puller</td><td>HEKA</td><td>Sutter P-97</td><td></td></tr><tr><td>Neurolucida system</td><td>Microbrightfield</td><td></td><td>with Neurolucida and Neuroexplorer softwares</td></tr></tbody></table>

electrophysiological and morphological characterization of neuronal microcircuits in acute brain slices using paired patch-clamp recordings

的膜片钳记录，从急性脑切片制剂与同时细胞内生物胞素填充两个（或多个）耦合突触神经元（配对记录）的组合允许它们的结构和功能特性的相关分析。用这种方法，可以通过它们的形态和电生理反应模式来识别和表征前和突触后神经元。配对录音允许研究这些神经元之间的连接模式，以及化学和电突触传递的属性。在这里，我们得到，以获得可靠的配对记录连同神经元形态学的最佳恢复所需的步骤的一步一步的描述。我们将描述如何对经由化学突触或间隙连接连接的神经元在大脑切片制剂被识别。我们将阐述如何神经元被重建，以获得dendrit自己的3D形态IC和轴突域以及如何突触联系的识别和本地化。我们还将讨论配对记录技术的警告和限制，尤其是脑片的制备过程中与树突和轴突截断相关，因为这些强烈地影响连接的估计。因为配对记录方法的多功能性。然而，它会留在在大脑中的神经元鉴定微电路表征突触传递的不同方面的宝贵工具。

配对录音是选择的方法进行了深入的表征形态识别的单向或双向的突触连接，以及间隙连接（电）连接（ 图1）。在体感桶皮质层4配对记录的一个例子示于图1A。两个单向兴奋性和抑制性突触连接可被表征（ 图1B中的C）。此外成对录音允许来自双向突触连接， 即 ，记录，从连接，其中两个神经元中的一对是前和突触后彼此。这示于图1D，<...

Watch this Scientific Journal Video about Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings at JoVE.com

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

神经微电路急性脑片电生理和形态特征采用配对膜片钳记录

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

electrophysiological-morphological-characterization-neuronal

Research

JoVE Journal

Neuroscience

17.2K 次观看.  Research Centre Jülich. Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

视频: Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

调查轻度创伤性脑损伤海马电路功能的改变

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

科研

神经科学

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

切片热门:成人急性脑切片在生理温度

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where the stimulus applied depends in real-time on the response of the system. Recent applications range from the implementation of virtual reality systems for studying motor responses both in mice1 and in zebrafish2, to control of seizures following cortical stroke using optogenetics3. A key advantage of closed-loop techniques resides in the capability of probing higher dimensional properties that are not directly accessible or that depend on multiple variables, such as neuronal excitability4 and reliability, while at the same time maximizing the experimental throughput. In this contribution and in the context of cellular electrophysiology, we describe how to apply a variety of closed-loop protocols to the study of the response properties of pyramidal cortical neurons, recorded intracellularly with the patch clamp technique in acute brain slices from the somatosensory cortex of juvenile rats. As no commercially available or open source software provides all the features required for efficiently performing the experiments described here, a new software toolbox called LCG5 was developed, whose modular structure maximizes reuse of computer code and facilitates the implementation of novel experimental paradigms. Stimulation waveforms are specified using a compact meta-description and full experimental protocols are described in text-based configuration files. Additionally, LCG has a command-line interface that is suited for repetition of trials and automation of experimental protocols.

Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board.

实时电:采用闭环协议来探测神经动力学和超越

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

用优化的n-甲基 d-葡乙胺保护恢复法制备急性脑切片

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

在脑片全细胞膜片钳记录

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze motor-learning induced plasticity, we trained rats using an accelerated rotor rod task. Rats performed the task 10 times at 30-s intervals for 1 or 2 days. Performance was significantly improved on the training days compared to the first trial. We then prepared acute brain slices of the primary motor cortex (M1) in untrained and trained rats. Current-clamp analysis showed dynamic changes in resting membrane potential, spike threshold, afterhyperpolarization, and membrane resistance in layer II/III pyramidal neurons. Current injection induced many more spikes in 2-day trained rats than in untrained controls.
To analyze contextual-learning induced plasticity, we trained rats using an inhibitory avoidance (IA) task. After experiencing foot-shock in the dark side of a box, the rats learned to avoid it, staying in the lighted side. We prepared acute hippocampal slices from untrained, IA-trained, unpaired, and walk-through rats. Voltage-clamp analysis was used to sequentially record miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) from the same CA1 neuron. We found different mean mEPSC and mIPSC amplitudes in each CA1 neuron, suggesting that each neuron had different postsynaptic strengths at its excitatory and inhibitory synapses. Moreover, compared with untrained controls, IA-trained rats had higher mEPSC and mIPSC amplitudes, with broad diversity. These results suggested that contextual learning creates postsynaptic diversity in both excitatory and inhibitory synapses at each CA1 neuron.
AMPA or GABAA receptors seemed to mediate the postsynaptic currents, since bath treatment with CNQX or bicuculline blocked the mEPSC or mIPSC events, respectively. This technique can be used to study different types of learning in other regions, such as the sensory cortex and amygdala.

The slice patch clamp technique is an effective method for analyzing learning-induced changes in the intrinsic properties and plasticity of excitatory or inhibitory synapses.

分层膜片钳技术在学习诱导可塑性分析中的应用

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

自动图像引导贴片夹在脑切片神经元研究中的应用

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Preparation of Acute Spinal Cord Slices for Whole-cell Patch-clamp Recording in Substantia Gelatinosa Neurons

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms underlying sensory transmission, nociceptive regulation, and chronic pain or itch development. Implementations of electrophysiological recordings together with morphological studies based on the utility of acute spinal cord slices have further improved our understanding of neuronal properties and the composition of local circuitry in SG. Here, we present a detailed and practical guide for the preparation of spinal cord slices and show representative whole-cell recording and morphological results. This protocol permits ideal neuronal preservation and can mimic in vivo conditions to a certain extent. In summary, the ability to obtain an in vitro preparation of spinal cord slices enables stable current- and voltage-clamp recordings and could thus facilitate detailed investigations into the intrinsic membrane properties, local circuitry and neuronal structure using diverse experimental approaches.

Here, we describe the essential steps for whole-cell patch-clamp recordings made from substantia gelatinosa (SG) neurons in the in vitro spinal cord slice. This method allows the intrinsic membrane properties, synaptic transmission and morphological characterization of SG neurons to be studied.

凝胶神经元全细胞斑块夹的急性脊髓切片的制备

Recent whole-cell patch-clamp studies from substantia gelatinosa (SG) neurons have provided a large body of information about the spinal mechanisms ...

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic multiplicity). Synaptic multiplicity is plastic and changes throughout development and in different physiological conditions, being an important determinant for the efficacy of synaptic transmission. Here, we outline experiments for estimating the degree of multiplicity of synapses terminating onto a given postsynaptic neuron using whole-cell patch clamp electrophysiology in acute brain slices. Specifically, voltage-clamp recording is used to compare the difference between the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). The theory behind this method is that afferent inputs that exhibit multiplicity will show large, action potential-dependent sEPSCs due to the synchronous release that occurs at each synaptic contact. In contrast, action potential-independent release (which is asynchronous) will generate smaller amplitude mEPSCs. This article outlines a set of experiments and analyses to characterize the existence of synaptic multiplicity and discusses the requirements and limitations of the technique. This technique can be applied to investigate how different behavioral, pharmacological or environmental interventions in vivo affect the organization of synaptic contacts in different brain areas.

Here, we present a protocol for evaluating the functional synaptic multiplicity using whole-cell patch clamp electrophysiology in acute brain slices.

全细胞膜片电生理方法对突触倍数的评价

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic ...