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。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Name</td>
			<td style="width:199px;">Company</td>
			<td style="width:199px;">Catalog Number</td>
			<td style="width:199px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Amplifier</td>
			<td style="width:199px;">HEKA</td>
			<td style="width:199px;">EPC 10 USB Triple</td>
			<td style="width:199px;">with 2-3 preamplifiers</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:199px;">Microscope</td>
			<td style="width:199px;">Olympus</td>
			<td style="width:199px;">BX51WI</td>
			<td style="width:199px;">with 2 camera ports and a 4&#215; objective, a 40&#215; water-immersion objective</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Camera</td>
			<td style="width:199px;">TILL Photonics</td>
			<td style="width:199px;">VX55</td>
			<td style="width:199px;">infrared CCD camera</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:199px;">Workstation</td>
			<td style="width:199px;">Luigs &#38; Neumann</td>
			<td style="width:199px;">Infrapatch 240</td>
			<td style="width:199px;">with a motorized x-y stage and a motorized focus axis for the microscope</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Micromanipulator</td>
			<td style="width:199px;">Luigs &#38; Neumann</td>
			<td style="width:199px;">SM-5</td>
			<td style="width:199px;">x-y-z manipulators for 2-3 preamplifiers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Faraday cage</td>
			<td style="width:199px;">Luigs &#38; Neumann</td>
			<td style="width:199px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Anti-vibration table</td>
			<td style="width:199px;">Newport Spectra-Physics</td>
			<td style="width:199px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Patchmaster</td>
			<td style="width:199px;">HEKA</td>
			<td style="width:199px;"></td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Microtome</td>
			<td style="width:199px;">Microm International</td>
			<td style="width:199px;">HM650V</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Micropipette puller</td>
			<td style="width:199px;">HEKA</td>
			<td style="width:199px;">Sutter P-97</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:199px;">Neurolucida system</td>
			<td style="width:199px;">Microbrightfield</td>
			<td style="width:199px;"></td>
			<td style="width:199px;">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

17.1K 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

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

从一个暗适应切片制备小鼠视网膜神经细胞的膜片钳录音

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

科研

神经科学

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

配对补丁钳从电机神经元和斑马鱼的目标骨骼肌

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike 1. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex 2. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative 3, 4: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening 5. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons 6; recently, we have also reported progress towards interrogating mammalian neurons 7. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel.The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
<img alt="Figure 1" src="/files/ftp_upload/3288/3288fig1.jpg" />
Figure 1. Principle of measurement using the NRCC patch-clamp chip
We detail here the protocols to sterilize and prime the chips, load them with medium, plate them with cells, and finally use them for electrophysiological recordings.

We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings.

NRCC的膜片钳芯片上细胞的培养和电

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

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

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

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

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

膜片钳电容测量和Ca 2 +单神经末梢成像在视网膜切片

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

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

膜片钳记录在内耳毛细胞的分离斑马鱼

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

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 ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

从胚胎斑马鱼的毛特纳细胞膜片钳记录

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

计算机辅助多电极膜片钳系统

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

在器官型海马脑片搭配全细胞记录

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

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 ...