作者要感谢达纳Kamalidenova，摩根Autexier，和罗克Chopier，谁做的视频和动画，以及弗洛里安·贝克的照片和后期制作的视频语音。这项工作是由奥林巴斯赞助和支持职业发展奖（HFSPO），ANR（规划署，青年chercheurs和神经科学计划相思），FRC（联邦争取RECHERCHE SUR LE Cerveau），INSERM和LaPitiéSalpêtrière医院（转化研究的补助金合同）NR，从法国研究部和德意志研究联合会博士后奖学金，并从学校的博士“生命科学中的前沿”，巴黎狄德罗大学，贝当古·舒勒基金会，FRM（基金会争取RECHERCHEMédicale）博士生奖学金到JS

1。的人工脑脊液和细胞内液的制备<ol><li>在实验开始前，需要准备的内部溶液的膜片钳记录，以及人工脑脊液（ACSF）海马准备。此外，您将需要一个解剖的套件，包括手术剪刀和精细虹膜剪，小铲和钳子（精细科学的工具），玻璃脱气装置（微过滤器的蜡烛，ROBU德国）和组织网格（蚊网或尼龙紧张的），以及强力胶（UHU登特）。芬克尔和书商，2001年1描述的的电片修补程序设置的配置。 </li><li>对于内部的解决方案中，溶解（以mM计）：105 K-葡糖酸，30氯化钾，10 HEPES和0.3 EGTA，在去离子水中（在最终体积的70％-80％）。溶液冷却至4℃，并添加（以mM计）：4的ATP-Mg，0.3 GTP-Tris和10磷酸肌酸。调整pH值至7.4与KOH和填充üp与去离子水至最终体积。 （Osmolaritiy：〜280毫渗）。过滤解决方案（孔径为0.2微米）。分装溶液是稳定的3-4周，在 - 20°C。对于一个实验天〜1毫升的内部溶液是必要的。 </li><li>除非另有说明，学联用于海马CA1区中的细胞的制备和录音，包含（以mM计）：119的NaCl，2.5氯化钾的CaCl 2，2.5，1.3用MgSO 4，1的NaH 2 PO 4，26.2的NaHCO 3和11葡萄糖。这些盐溶解在去离子水中（渗透压〜320毫渗）和含氧此解决方案与Carbogen增（95％O 2和5％CO 2）的至少10分钟（pH值〜7.3 - 7.4）。准备每个实验至少1升的溶液。应特别注意准备海马组织将被用来进行实验的海马CA3区。事实上，这个区域是容易出现癫痫样活动和随后的神经元死亡。因此，SYNAPT应大大减少集成电路活动切片的制备过程中，这是通过执行海马解剖冰冷的蔗糖溶液中含有（以mM计）：87 0.5 2.5氯化钾，氯化钠，氯化钙2，7的MgCl 2，1的NaH 2 PO 4的 ，10 碳酸氢钠 ，葡萄糖和75蔗糖25日。在这个解决方案中，低钠，低血钙和高浓度的镁相结合，大量减少突触前发射和释放的可能性，以及突触后NMDA受体的活性，从而减少自发活动和细胞死亡。一旦准备，海马脑片CA3区的细胞从录音用于，灌流改性学联，含有4mM的的CaCl 2和4用MgSO 4，以尽量减少多突触活动。 </li></ol> 2。急性海马切片制备的<ol><li> 〜300毫升的学联切片室，以及用于制备在4℃下冷​​却下来，同时不断充氧Carbogen增。准备一个小烧杯中，在室温（RT）与学联切片存储Carbogen增（计划图1），这也是含氧。 </li><li>发动机罩下，用小纸巾浸泡入笼中加入用1毫升异氟烷麻醉鼠标。 </li><li>后鼠标是深度麻醉，切头，将其直接添加到一个小盘子，用冰冷的充氧ACSF。卸下头皮用小剪刀和随后的步骤，转移到组织的头。开始解剖海马，作为在图1中示出和描述。 </li><li>剪切300-400微米厚的横切片在低转速（3微米/秒）和振动频率为70赫兹，在冰冷的含氧学联，并将其传送到一个​​存储室。让切片其余在RT事先记录为至少1小时。切片为CA3实验的保存在34°C和25分钟，在室温下至少30分钟，从切片过程中恢复。 </li></OL&gt; 3。双重录制诱发星形胶质细胞电流和神经元的电位在这里，我们描述了如何记录突触诱发星形胶质细胞和神经元的反应， 即通过使用一个外电极突触激活通过afference刺激反应。 <ol><li>不断灌注录音室，与充氧ACSF（1.5-2毫升/分钟，RT），含有100μM印防己毒素（GABA A受体拮抗剂）隔离兴奋反应的。切片转移到聚-L-赖氨酸涂布盖玻片（1.5至3毫克/毫升）中，浸泡的液体，实现了良好的切片粘附和切片上添加一个的ASCF下降。放置到记录室盖玻片。封锁抑制 ​​传输印防己毒素可导致癫痫样活动， 即自发的，同步触发的神经元群体，这将扭曲的测量诱发的事件。因此，为了防止癫痫样活动，使CA1和CA3区的平砍（只是表面上的）之间通过Schaffer侧支（ 图2a），以防止传播。 </li><li> 地层的radiatum星形胶质细胞可以通过他们的小胞体尺寸（〜10微米）和星状的过程组件。选择的单元格的片表面以下至少20-30微米。玻璃安装刺激电极（针尖电阻〜1MΩ），它连接到的刺激隔离框，并接地，以浴（简单地通过包装的周围的玻璃吸移管的第二银线）上的银线。 Schaffer侧支区域的刺激电极放置到，如在图2A中所示，在距离为200〜300微米的距离选择的星形胶质细胞。到氯化银线连接到探头放大器的安装现场记录电极（2-5MΩ）。这两个电极填充之前，用ACSF。选择在multiclamp模式I = 0，禁止外部com命令输入和放置电极远离到地层radiatum区域星形胶质细胞（ 图2A）〜50微米。记录响应与增益2到10和过滤器与一个2千赫贝塞尔滤波器。触发电流注入到大脑切片周围的Schaffer侧支轴突和随后的递质释放的突触前神经末梢动作电位的项目突触后CA1锥体神经元。被释放的发射器将触发通过突触后的离子型受体进入细胞内的正电荷的流动，这是可测量的细胞外作为一个小的负电位。此场兴奋性突触后电位（fEPSP的）结合的基团的活性，同时激活的神经元，药理学上被阻塞而抑制传输。应用一些测试脉冲（持续时间为0.1毫秒），以唤起一个fEPSP的一些重新定位的刺激电极可能有助于增加fEPSP的。一个典型的CA1 阶层放射这个fEPSP的， 如图2B所示。可以发现在进一步的细节上的定位响应波形和Yuan 等人，2003年2。 fEPSP的幅度通常应该在一个健康的海马脑片以上的纤维抽射幅度的两倍大。 fEPSP振幅的准确定量或斜坡，诱发的反应“应该是单突触，作为多突触活性（作为多峰值响应检测）表示独立的电刺激，这可能​​是一个标志为过度兴奋突触活动。对于海马CA3区中进行的实验，刺激和记录移液器被定位，如在图3C中示出。为了清楚地识别苔藓纤维输入，强烈促进，双脉冲刺激（50毫秒脉冲间隔）和1赫兹刺激几秒钟被施加到大量提高最初低振幅诱发fEPSP的responses.At到底的实验，DCGIV，一个mGluR2 / 3受体拮抗剂，可水洗，以进一步验证，确实苔藓纤维投入刺激。 〜90％，主要由于mGluR2 / 3受体，抑制突触前膜释放的苔藓纤维终扣的高表达，这拮抗剂的应用应减少fEPSP的。 </li><li>填充一个补丁吸移管（〜2-5MΩ）内部溶液过滤并装入它的氯化银线连接到第二探头上，用一个注射器中，这是通过连接管连接到您的吸液管保持施加正压。不断应用20毫秒为10 mV的测试脉冲，使移液管进入组织，直到到达细胞表面和膜中的偏转变得可见。的吸移管偏移归零，删除正压力，并夹持膜 - 80 mV的。等待直到一个gigaseal的（至少1GΩ）就达到了（它不应该需要更长的时间超过几秒钟）。柔和的应用程序的一些负面压力可能有助于实现公司igaseal。额实现进入细胞内的负压由一个短的应用，或使用快速切换在multiclamp功能的。同时启动记录Schaffer侧支诱发fEPSP的钳位电压（VHOLD - 80毫伏，频率0.1 Hz，2 kHz的贝塞尔滤波器，增益10）和星形胶质细胞反应。星形胶质细胞的反应是双相的：首先，你会看到一个快速的瞬时外向钾电流，反映相邻锥体细胞所产生的fEPSPs。这之后是缓慢上升的和衰减的内向电流（持续存在的几秒钟（&gt; 10秒）神经元的反应终止后）。此电流主要是由于钾进入星形胶质细胞，释放后通过周围去极化的突触后端子。同时启动了快速瞬态谷氨酸转运体（GLT），所引发的突触前谷氨酸释放被屏蔽的钾电流。保持电位的星形胶质细胞，以及接入电阻的补丁应是莫nitored在整个实验过程和变化不应超过20％，以避免由于拍摄条件的变化，星形胶质细胞反应变量不准确的监测。只有星形胶质细胞的最初持有潜力&gt; -70 mV的，应当进行调查，以研究健康细胞。 </li><li>切换电压至电流钳，以便记录的诱发的星形细胞膜的去极化。隔离灌注离子型谷氨酸受体拮抗剂犬尿喹啉酸（5毫米），胶质细胞谷氨酸转运体（GLT）完全封锁，直到fEPSP的和GLT幅度的已经达到了高原。要清楚地确定的GLT当前，应用特异性拮抗剂DL-苏-β-Benzyloxyaspartatic的酸（DL-TBOA的，200μM）。增加刺激强度的2倍〜5倍，以记录在不同的突触强度的神经细胞和星形胶质细胞的反应，和应用两个紧密间隔的刺激（50毫秒时间间隔），调查反应配对脉冲刺激。 R系列的稳定性抗性的神经胶质细胞和膜电位的期间应监测记录。 </li><li>为了可视化的程度的间隙连接介导的星形胶质细胞的网络，应染料耦合实验在电流钳模式下进行，没有任何电流注入的情况下，以使染料分子的低（&lt;1.5 kDa的），如磺基若丹明-B的被动扩散，通过间隙连接通道。为了尽量减少染料外溢到周围组织，正压应通过膜片微电极刚刚进入组织时，应尽快达成的补丁。 </li></ol>

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没有利益冲突的声明。

双记录的突触诱导的神经元和神经胶质细胞的反应是一个非常有用的方法，，网上学习改建前和突触后活动相关的星形胶质细胞特性的变化。突触诱发的神经胶质膜的去极化是一种直接测量细胞外钾离子的上升，部分由于突触前动作电位射击，但主要是突触后去极化7。因此录音的胶质膜电位的动态可以使用调查修改的兴奋性突触，突触后活动，细胞外空间的容积和钾的缓冲能力6，8。星形胶质细胞谷氨酸转运体电流的突触前谷氨酸的释放，能够监测短期变化，释放概率3，5，9是一个敏感的措施。在不同的突触或在不同发育第一，它可进一步被用来表征的功能性突触神经胶质细胞的相互作用10岁。应该强调的是GLTs温度敏感性高的11和被驱动的Na +，K +和H 12的电化学梯度。因此，振幅和动力学的GLT当前的高度依赖于所选择的实验条件。此外，星形胶质细胞的谷氨酸间隙过程中来自记录的GLT电流的实际的时间是已知的部分变模糊。这是由于过滤GLT电流的因素，如电张性能的星形胶质细胞，或异步递质释放，歪曲其动力学13。方法提取的过滤机制的时间的功能已被开发，并可以使用，以获得实际的谷氨酸间隙时间过程在生理或病理情况下，作为recenly执行6,13,14。此外，在电流钳，同时记录的星形胶质细胞的细胞膜去极化，可以提供INSIG高温超导可能改变细胞外钾瞬变。单星形胶质细胞联系起来，以10〜100种不同的神经元突触，因此整合和调整本地的神经元网络活动。 当使用这里介绍的技术， 即记录电全细胞的反应从星形胶质细胞为基础的突触活动中获得的见解，一个人应该记住，在星形胶质细胞，膜片钳记录在SOMA水平允许检测电流主要源于胞体或近端的过程。事实上，胞体检测到的电流只有部分源于精细的远端过程时激活的受体和渠道发生在多道精细的工序，可以产生强大的电流，传播到胞体。因此，基础受体和渠道活动覆盖突触车厢的个人的小星形胶质细胞过程是很难检测到。这是部分原因是由于有限的卵的胶质和时间控制膜电流和电压的全细胞膜片钳记录，从星形胶质细胞原位 。然而，应该注意的是，表面的大量微小的星形细胞进程超过迄今为止胞体和主要过程的膜面积。此外，这些perisynaptic的星形胶质细胞的微区包含功能相关的受体和渠道，这可能在的神经胶质通信和突触的调控中发挥了重要的作用。因此，我们这里介绍的是该技术主要用于研究神经元集合的同步活动，特别是在发生afference刺激的星形胶质细胞融合。它不应该被用于研究个人星形胶质细胞突触和相邻的精细过程发生在基础的自发活动之间的对话。研究当地的基础突触活动的星形胶质细胞反应的另一种方法是执行膜片钳记录道精细的工序，为d树突15。虽然修补这些精美的星形胶质细胞的过程可能具有挑战性，由于其体积小，它可能是一个途径，追求解开星形胶质细胞的微区之间更亲密的对话和个体突触。然而，可能小电星形胶质细胞反应产生的个人精美的星形胶质细胞的过程可能会低于阈值检测，电气噪声达到平均3-5 pA的膜片钳记录。研究星形胶质细胞的突触活动的另一种方法是钙成像，因为激活的星形胶质细胞的细胞膜受体或转运神经活性物质可以引起细胞内钙瞬变。然而，批量加载的星形胶质细胞钙指标主要反映躯体活动16。结合电生理和钙成像，也可以检测的小钙信号从细小的星形胶质细胞的过程，无论是自然发生的或触发bŸ最小的突触刺激17，18。然而，人们应该牢记，高亲和力的的钙指标可能像钙的缓冲区，抑制重要的钙信号转导通路，而低亲和力的指标可能低于检测水平。最后，一​​个优雅的和非侵入性技术研究钙事件，精致的星形胶质细胞的过程，这也规避冲刷的细胞内信号分子，在全细胞膜片钳，包括使用膜有针对性的钙传感器，它可以在星形胶质细胞中表达在原位 ，以及在体内 19。然而，钙成像只能提供一个信号分子，参与了许多，但并不是所有的细胞活动有关的信息，而全细胞膜片钳提供所有不同的离子电流通道和受体激活后引发的定量信息。因此同时从神经元和星形胶质细胞的电生理记录是一个独特而强大的在线神经胶质细胞离子的信号，它的作用大脑信息处理的动态方法解开。

<table border="1"><tbody><tr><td>的试剂的名称</td><td>公司</td><td>目录编号</td><td>评论（可选） </td></tr><tr><td>印防己毒素</td><td>西格玛</td><td> P1675 </td><td>溶于DMSO </td></tr><tr><td>犬尿喹啉酸</td><td> TOCRIS </td><td> 0223 </td><td>溶解在34℃下搅拌或超声</td></tr><tr><td> DL-TBOA的</td><td> TOCRIS </td><td> 1223 </td><td> DCG IV TOCRIS 0975 </td></tr></tbody></table>

dual electrophysiological recordings of synaptically-evoked astroglial and neuronal responses in acute hippocampal slices

星形胶质细胞与神经元的三方突触形成合力，在那里他们整合和调节神经元的活动。事实上，星形胶质细胞感测通过其的离子通道和神经递质受体激活的神经元的输入，并通过活性依赖性释放gliotransmitters部分的过程信息。此外，星形胶质细胞谷氨酸构成的主吸收系统，有助于钾空间缓冲以及GABA的间隙。因此，这些细胞不断监测突触活动，从而改变突触释放的谷氨酸，GABA和细胞外钾水平的敏感指标。此外，星形胶质细胞摄取活性的改变或缓冲能力产生严重影响神经功能，特性时，可能会忽略病理生理情况或基因敲除小鼠。双记录的神经元和星形胶质细胞的活动，因此是一个重要的方法来研究改变相关的伴随突触强度的变化，星形胶质细胞的吸收和缓冲能力。在这里，我们将介绍如何准备海马脑片，如何识别地层radiatum星形胶质细胞，并同时记录神经元和星形胶质细胞的电生理反应。此外，我们将介绍如何药理突触诱发的星形胶质细胞电流隔离。

一位代表同时记录突触诱发的星形胶质细胞和神经元的反应（fEPSPs）的海马CA1区中的图2中AB所示。诱发星形胶质细胞电流是双相的，即它是由一个瞬时外向钾电流和缓慢衰减的内向电流（&gt; 10秒）（ 图2B）。外向钾电流反映了诱发fEPSP的，被抑制的离子型谷氨酸受体阻断后，犬尿喹啉酸（暗灰色轨迹， 图2B）3。缓慢的内向电流的大部分反映钾进入星形胶质细胞的以下突触后去极化，因为它也废除了犬尿喹啉酸，抑制突触后离子型谷氨酸受体活性（ 图2B和图2C 1）3-6，已知以表示主源（80％）的钾离子的释放7。其余的迅速上升ð腐烂的内向电流抑制GLT拮抗剂DL-TBOA（浅灰色轨迹， 图2b和图2c）。事后减法从犬尿喹啉酸（深灰色迹线）中的电流的其它慢电流在TBOA（浅灰色轨迹）允许纯的星形胶质细胞谷氨酸转运体电流（黑色的迹线），如在图2C 2所示的隔离。持久慢慢衰减电流犬尿喹啉酸和TBOA（浅灰色跟踪， 图2B和图2C 2）可以被阻塞者TTX（数据未示出），并反映最有可能积累的细胞外K +过程中释放的突触前传入神经 ​​烧成3。中度单一刺激Schaffer侧支诱导一个相对大的突触诱发的星形胶质细胞相比小诱发记录在相同的细胞（ 图2D）的去极化电流。这是由于低的膜电阻的星形胶质细胞。记录的突触诱发的星形胶质细胞的膜电位的动态，2D图所示，是一个直接测量局部细胞外钾水平8。诱发的星形胶质细胞的反应相关的神经元的电活动的正常化允许直接比较不同的实验，最近显示6。星形胶质细胞的电流还可以非常可靠地监测兴奋传导的改变，作为总的突触诱发的星形胶质细胞的电流如下线性在fEPSP的增加（ 图2E）。神经元，星形胶质细胞的电流也反映了短期突触可塑性的，因为它们表明，双脉冲便利（ 图2F）。配对的CA1区锥体细胞和星形胶质细胞的全细胞记录揭示的两种类型的细胞中，由于神经元的显示在响应一个去极化脉冲的动作电位的非常不同的电行为，而相邻的星形胶质细胞（ 图3A-B）是沉默。然而，中度刺激Schaffer侧支能唤起同时快速的兴奋posynaptic CA1区锥体细胞的潜力和快速的向外和缓慢的内向电流在相邻的星形胶质细胞（ 图3B 2）。双录音突触诱发的神经细胞和星形胶质细胞的反应也可以被记录在海马CA3区，如在图3C中示出。事实上，单CA3区苔藓纤维的刺激唤起的基础条件非常小的神经元的反应，记录fEPSPs，相关的小，速度快，向外和慢内向电流的星形胶质细胞（ 图3D）。相反，1赫兹CA3区苔藓纤维为几秒钟的刺激强烈会加强其fEPSP的，而它的星形胶质细胞的反应（ 图3D 2）只有适度增加。 图1。海马隔离，以准备横向切片。要解剖的大脑，切断的橹沿中线（一）。品牌的冠切在嗅球的水平（二），并随后在小脑（三）的水平。小心地取出橹的钳子（D）的帮助下，用刀片（E）分开的两个半球，并将它们传送到一小汤匙冷氧合学联（F）。 〜5分钟，平衡后，干纸巾上放置一个半球的内侧表面向上（G）。随着两匙的帮助下，取出间脑（HJ）。海马现在是可见的，如图所示的虚线（十一）。解剖海马，用勺子，从伞，可见为白色结构（LM）。转移海马回冷学联。准备一个小的琼脂糖凝胶块，位置的alveus两个海马和腹侧海马我们面临的琼脂块的边缘，和浸泡仔细所有液体程允许琼脂（n）的一个很好的附件。胶的海马腹侧部（O）的琼脂块上。 <img src="/files/ftp_upload/4418/4418fig2.jpg" alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/4418/4418fig2highres.jpg"/> 图2。同时反应诱发的海马CA1区突触在神经元和星形胶质细胞。 A）的计划的海马切片，示出的布置的刺激电极，以激活Schaffer侧支（SC），该补丁电极内液，记录星形细胞电流，和细胞外电极，记录fEPSP的，由SC刺激诱发的海马CA1区。 B）代表同时录音的fEPSPs（上面板）和星形胶质细胞的电流（下面板）由SC在presenc刺激诱发突触的痕迹E的药理药物。首先记录中存在的GABA A受体阻断剂（印防己毒素，100μM，黑色痕迹）来隔离兴奋反应的反应。后续的应用离子型谷氨酸受体阻断剂（犬尿喹啉酸，5毫米，暗灰色的痕迹），抑制fEPSP的和持久的星形胶质细胞电流的主要部分，揭露一个小型，快速的暂态分量的星形胶质细胞的电流响应，这是敏感的谷氨酸的转运受体阻滞剂（TBOA，200μM，浅灰色的痕迹）。星形胶质细胞15 pA的电流，比例尺，fEPSP的0.1毫伏，10毫秒。 C 1）。示例跟踪的星形胶质细胞的钾电流（1-2），它可以从诱发反应，在B（下面板，黑色痕量）所示，分离减去残留在犬尿喹啉羧酸（2）的总电流的电流分量（ 1）。比例尺，1秒，20 pA的。的C 2）有样品一丝谷氨酸转运电流（2-3），通过以下方式获得减法TBOAınsensitive缓慢从犬尿喹啉羧酸（2）中的电流（3）成分。比例尺，2.5 Pa时，25毫秒。 D）记录的电压钳（下面板）和记录在相应的膜的去极化电流钳（上面板）由SC刺激的星形胶质细胞中诱导的向内的电流的示例痕迹。比例尺，电流 - 电压钳5 pA，则1秒1.5 mV时，钳位。 E）示出的突触前纤维截击（输入）和星形胶质细胞的总电流（输出）之间的关系的输入输出曲线同时记录响应到SC刺激组（n = 6）。电流的增加而线性增加的纤维截击星形胶质细胞，神经元fEPSP的。 F）样品在40毫秒脉冲间隔的双脉冲刺激神经元的反应（fEPSP的）和星形胶质细胞电流的痕迹。突触诱发的星形胶质细胞的展品，如神经元，双脉冲便利。 5 PA，比例尺为0.1 mV，20毫秒。 图3。双录音的海马CA1和CA3区突触诱导的神经元和星形胶质细胞反应。 A）重建的CA1锥体细胞注射用磺基罗丹明B（红色，0.1％）和星形胶质细胞填充用荧光素葡聚糖（绿色，0.1％），采用全细胞膜片钳技术。比例尺，10微米。 B 1）代表同时从CA1锥体细胞和邻近的星形胶质细胞的膜电位记录在电流钳全细胞记录的痕迹。诱发的神经元动作电位的烧成（​​黑色痕量），通过注射20 pA的去极化脉冲电流，唤起在相邻的星形胶质细胞（灰色痕量）没有响应。 10 pA的比例尺，20 mV时，100毫秒。 B 2）双通道全细胞记录的当前蚌CA1锥体细胞样本的痕迹p和SC印防己毒素（100μM）存在下，在刺激后的电压钳位在相邻的星形胶质细胞。 SC刺激唤起的兴奋性突触后电位（EPSP，黑色痕量），相关联的一个小的和持久的星形细胞电流（灰色痕量）。 10 pA的比例尺，5毫伏，100毫秒。 C）计划的海马脑片描绘海马齿状回（DG），CA3区的安排，刺激电极，以激活苔藓纤维，膜片微电极电极（灰色），记录星形胶质细胞的电流，和外电极， fEPSP的记录，CA3区苔藓纤维刺激诱发的。 D，E）代表的痕迹配对录音的CA3 fEPSPs（上部面板，在D 1和D 2中的黑色的痕迹）和星形胶质细胞的全细胞的反应（低级面板，在D 1和D 2中的灰色的痕迹）CA3生苔单一刺激0.02赫兹（放大中的插图）（D 1）的纤维，或在1 Hz刺激频率（D 2）。比例尺为D <sub&gt; 1和D 2，为0.2 mV，15 pA的，时间：1秒，D 2 100毫秒。插入比例尺，0.1 mV时，100毫秒。

Watch this Scientific Journal Video about Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices at JoVE.com

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

编制急性脑切片中分离出海马，以及同步的星形胶质细胞和神经元的电生理记录<em&gt;地层radiatum</em刺激Schaffer侧支时。隔离的星形胶质细胞的钾和谷氨酸转运体电流的药理证明。

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 ...

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27.1K 次观看.  Collège de France. 编制急性脑切片中分离出海马，以及同步的星形胶质细胞和神经元的电生理记录地层radiatum

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

Dual Somatic Recordings from Gonadotropin-Releasing Hormone (GnRH) Neurons Identified by Green Fluorescent Protein (GFP) in Hypothalamic Slices

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These gonadotropins are essential for the regulation of reproductive function. The GnRH-containing neurons are distributed diffusely throughout the hypothalamus and project to the median eminence where they release GnRH from their axon terminals into the hypophysiotropic portal system (1). In the portal capillaries, GnRH travels to the anterior pituitary gland to stimulate release of gonadotropins into systemic circulation. GnRH release is not continuous but rather occurs in episodic pulses. It is well established that the intermittent manner of GnRH release is essential for reproduction (2, 3).
Coordination of activity of multiple GnRH neurons probably underlies GnRH pulses. Total peptide content in GnRH neurons is approximately 1.0 pg/cell (4), of which 30% likely comprises the releasable pool. Levels of GnRH during a pulse (5, 6), suggest multiple GnRH neurons are probably involved in neurosecretion. Likewise, single unit activity extracted from hypothalamic multi-unit recordings during LH release indicates changes in activity of multiple neurons (7). The electrodes with recorded activity during LH pulses are associated with either GnRH somata or fibers (8). Therefore, at least some of this activity arises from GnRH neurons.
The mechanisms that result in synchronized firing in hypothalamic GnRH neurons are unknown. Elucidating the mechanisms that coordinate firing in GnRH neurons is a complex problem. First, the GnRH neurons are relatively few in number. In rodents, there are 800-2500 GnRH neurons. It is not clear that all GnRH neurons are involved in episodic GnRH release. Moreover, GnRH neurons are diffusely distributed (1). This has complicated our understanding of coordination of firing and has made many technical approaches intractable. We have optimized loose cell-attached recordings in current-clamp mode for the direct detection of action potentials and developed a recording approach that allows for simultaneous recordings from pairs of GnRH neurons.

Dual Somatic Recordings from Gonadotropin-Releasing Hormone GnRH Neurons Identified by Green Fluorescent Protein GFP in Hypothalamic Slices

Activity in neuronal systems often requires synchronous action potential discharges from neurons within a specific population. For example, pulses of gonadotropin-releasing hormone (GnRH) likely require coordinated activity between GnRH neurons. We present our methodological approach for reliably obtaining simultaneous electrophysiological recordings from the diffusely distributed GnRH neurons.

双体录音从促性腺激素释放激素（GnRH）神经元下丘脑薄片鉴定绿色荧光蛋白（GFP）

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating ...

科研

神经科学

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 ...

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

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

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 ...

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.

This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.

使用急性海马脑片啮齿动物突触标记/捕获和交叉捕获的研究

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity ...

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

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

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

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

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

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

急性<em&gt;体内</em&gt;麻醉大鼠超导途径局部场电位和多单位活动的电生理记录

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

海马鼠脑切片的突触密度评价

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Optogenetic 在行为小鼠海马θ振荡中的作用

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

为电子生理记录准备急性人类河马切片

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.