这项工作是由瑞典研究理事会，瑞典乌普萨拉大学和弗雷德里克和英格丽Thurings基金会。钧从EXODIAB和瑞典语Sällskapet为Medicinsk Forskning举行了博士后。我们感谢李敬技术援助。

1。脑片的制备: 海马脑片从产后16 - 22日龄Wistar大鼠进行实验。 <ol><li>按照当地的道德准则和批准的动物保健协议断头处死动物。 </li><li>迅速取出大脑用抹刀和削减沿中线用手术刀片（＃24）。广场上的Whatman滤纸每个半球所面临的削减纸张。 </li><li>把标本磁盘上的强力胶下降，切面朝下半球的地方在它的上面。将试样切割室充满冰冷的人工脑脊液vibratome磁盘（学联，请在这里看到低于3.1）。 </li><li>设置切片厚度为400微米，并开始切割大脑。您将需要您到达海马之前切断对脑组织的约2毫米。 </li><li>海马被放到一个玻璃培养皿填充板与学联。该板块是放置在一个黑色的背景（如黑纸），允许容易可视化的海马。用锋利的手术刀从周围组织分离的海马。要小心，不要推或拉的组织。 </li><li>切片，然后孵育° C为1小时，然后在常温下保存，直到实验完成37.0学联的解决方案。在一个小时的孵化组织恢复从切削过程中所施加的损害。 </li></ol> 2。用于修补吸液管的制备: <ol><li>移液器是由高硼硅玻璃毛细管。 </li><li>补丁移液器在吸液管上的自动或手动移液器车夫拉着的电阻2 - 4MΩ全细胞记录或8 - 20MΩ单声道录音时充满适当的电极内液和液学联（见下文3.1）。 </li><li>我们为单声道录音使用的移液器使用microforge坐在铂丝熔化玻璃的热量使表面平滑抛光。相同的高硼硅玻璃是在补丁的吸液管，用于线路上的玻璃液滴的形式。抛光的移液器在我们的经验，这种方式创建，形成更加稳定和较高的耐药性的密封比未涂层线热或打磨抛光步骤在自动拉拔移液器移液器。移液器的最后尺寸范围从8到20MΩ。全细胞记录中使用的移液器不抛光。 </li></ol> 3。在实验中使用的解决方案: 具体的解决方案是用于单通道和全细胞记录。 <ol><li>学联包含毫米:124氯化钠，26 3碳酸氢钠3，氯化钾，1.3，2.5 硫酸镁 NA 2 HPO 4，2.5氯化钙 ，10血糖与pH值7.2 - 7.4时，95％O 2和5％的CO 2冒泡。学联有时也辅以其他营养物质，如乳酸11。 </li><li>单声道录音: 洗澡的解决方案是学联或（毫米）:140氯化钾，氯化钠，5 2 1氯化镁，1.8 氯化钙和10工商业污水附加费（的N -三羟甲基]甲基-2 -氨基乙磺酸）pH值7.25和其他药物可能需要对具体的实验140胆碱氯，5氯化钠，1氯化钾，氯化镁，1.8氯化钙，pH值7.25 10工商业污水附加费，200μMsaclofen吸管的解决方案:在MM细胞贴附式和内而外的补丁。 （GABAB受体拮抗剂）和0 - 氨基丁酸1μM。对于外界的补丁（毫米）:140 NaCl或胆碱氯，0.3氯化钾，2，0.5 MgCl 2的氯化钙，5 EGTA 10工商业污水附加费，pH值7.2 - 7.4 。 </li><li>全细胞电压钳录音: 洗澡的解决方案 :切片灌流（2 - 3毫升/分）现在包含与学联也kynurenic酸（3毫米）和其他药物，可能需要具体的实验液中包含（毫米）:140 CSCL，1氯化钙2，3 EGTA，0.5氯化钾，氯化镁2，2个ATP镁，0.3 GTP钠，溴化5 QX - 314，10工商业污水附加费7.25 pH值与CsOH 。 </li></ol> 4。成型膜片钳实验的一个千兆欧姆（GΩ）封: 海马脑片（图1A）在录音室的底部由一个U形的铂丝上的尼龙线。为了形象化如组织CA1区和3个组织和海马或特定的细胞的齿状回区域，你需要一台显微镜。大多数实验室使用从10X到60X从而使识别地区或特定的​​细胞组织的目标一个堂堂正正的显微镜。然而，即使是解剖镜下修补片可以用来然后您直观地确定一个地区的补丁，但没有一个特定的细胞。这种类型的修补被称为"盲目补丁"。我ñ这两种情况下，到底是修补一个单细胞在特定的区域。成功率是两种方法类似。虽然在海​​马的主要细胞的细胞体海马的所有子域和阶层之间的高度结构层分散（例如CA3/CA1阶层pyramidale确定为明亮的乐队在海马脑片）举办抑制细胞的机构interneurons，弥补约10％的神经总人口12。偶尔中间神经或神经胶质细胞，颗粒细胞或锥体神经元层，可修补，可分化的主要细胞，这些细胞由不同的电气特性（见海马图书13 ）。 <ol><li> GΩ的密封形成。在这个过程中，玻璃吸管，从某种程度上成为紧密连接到细胞膜，并在不同的补丁配置（请参阅下文，4日和5），我们可以衡量GABA激活细胞膜的渠道活动。 吸管放入吸管持有人，并拧紧瓶盖，系移液器。 Axoclamp放大器200B上的设置是:V型钳，增益2，过滤器2 kHz时，保持电位为0 mV和我们正在做实验，通过使用PClamp软件的计算机。 </li><li>一块是连接到连接到吸管持有人，并关闭阀口片和管之间的管口吹入。这将创建在您的吸管正压。正压移液器有两个功能:第一，流出来的移液器和移液器一次进入浴缸的解决方案，这将有助于保持枪头清洁。二，通过补丁的吸管轻柔的吸力细胞膜的重要组成部分，是形成了一个高品质，高电阻密封。密封往往可以简单地打开阀门，控制吸管压力形成的。正因为如此，要确保有不漏气。移液器持有人，你可以很容易泄漏测试，浸成一个烧杯中放入口中片充满水和打击。如果你看到任何气泡逸出，那么这部分是不够紧。 </li><li>进液下您的吸管。在计算机屏幕上，你的吸液管电阻测量，你会看到一个正方形5 mV的脉冲序列和一个数字，告诉您您的吸液管的阻力产生的脉冲。较小的数字代表了较大的枪头开口，较大的移液器。调整偏移为0的吸管。 </li><li>密封的细胞膜。确定您要修补的区域或细胞，也就是形成一个密封。在大脑切片，可以识别的齿状回颗粒或明亮的条带海马CA1和CA3锥体细胞身体部位（见图1A）上使用的显微镜​​10X放大倍率或使用，我们可以找出个人锥体神经元表面60X的放大倍率。 <ol><li>纳入重点吸管，让您看到您所在地区/细胞和吸管，当你看，通过显微镜观察眼件。 </li><li>将上述区域/细胞，使用粗运动操纵者设置和降低吸管中间的吸管，直到它的正上方，但不接触的组织。 </li><li>到您所在地区/细胞中，轻轻地降低您的吸管使用您的机械臂上的精细运动的设置，并在同一时间观看计算机屏幕上，并跟踪您的吸液管电阻。保持你的吸管向下移动，直到你看到的吸液管的阻力越来越大（高数）。 </li><li>现在，轻轻地打开阀门释放正压移液器。这一个温柔的吸力，往往一个千兆密封（高阻抗密封，GΩ）形式automatically.If的金额，然后应用吸口的一块，你以前应用正压和千兆密封形式。 </li><li>现在，您已经获得了所谓的细胞贴附式膜片钳配置（见图1B） 。调整快，慢电容补偿取消从吸管和吸管持有人的瞬变。在计算机屏幕上，你将看到一个单位的当前跟踪。更好的信号噪声比GΩ的密封值越高，你会在单声道录音。 </li></ol></li></ol>它有时是有利补丁的海马脑片内。在某些情况下，细胞能健康的不是表面上时，为他们更多的保护。此外，在研究细胞功能的细胞外环境的影响，如外GABA的浓度滋补作用在神经元内，修补组织的目前这一代，可能是一种优势。修补程序，一个除了上面所述的相同，不会释放蒲片内，直到枪头移液器sitive压力。 5。建立全细胞配置和记录全细胞电流: <ol><li>在细胞连接的配置，改变吸管潜力-60毫伏或接近你的细胞的静息膜电位。 </li><li>吸吸管，通过管连接到吸管持有人，并在同一时间观看计算机屏幕上，注意在电阻和电容瞬变出现，一旦你已经进入细胞打破的变化。当发生这种情况，你在所谓的全细胞膜或全细胞模式（图1B） 。 细胞膜的电阻变化，从一个细胞类型到另一个取决于在膜的渠道和多少是开放的电导。细胞膜电容随细胞的大小。吸最初应轻轻，但如果它是很难打入细胞更多的力量可以应用。也可以用吸的脉冲。 </li><li>有时一个电阻补偿，吸管和细胞之间的发展是必需的。这就是所谓的系列或接入电阻（RS或RA）的赔偿，并可以通过软件或使用放大器上的控制旋钮手动。在我们的实验中，我们不补偿RA，但实验被拒绝，如果它是由25％以上的变化，从最初的接入阻抗。 </li><li> GABA激活进补，在全细胞模式记录电流。 </li></ol>在海马CA1区锥体细胞，我们在整个​​单元配置设置在控股的潜力 - 为60 mV，等待5 - 10分钟，使电极内液与细胞内部达到平衡。在这段时间录制的稳定。在实验过程中，我们不断跟踪串联电阻值。 实验是在电压钳和你衡量人口通过多种渠道在全​​细胞膜电流。一个可以区分突触和突触外GABA激活电流，电流的相位和滋补性质。拮抗剂SR95531抑制所有GABA的一个电流。控股目前的下降表明，目前在神经元的GABA激活进补水平。 正如我们感兴趣的是在切片中的依赖外，环境γ-氨基丁酸的补品GABA激活电流，我们通常不打补丁的细胞表面上，而是，片内的细胞。激动剂和拮抗剂，我们使用来调节A通道是在洗澡的解决方案的应用GABA和片内到达细胞。在试验室的解决方案流的速度是2 - 3毫升/分钟。 6。记录单声道电流: 实验是在细胞贴附，里面出或外膜片钳配置（请参见下面），并通过单一通道的分子，我们记录电流，单通道电流。 Axoclamp 200B型，V型夹，增益500，过滤器2或5千赫的设置。随着PClamp软件控制的吸管潜力，设置不低于100微秒的数据采样率和记录电流。为了尽量减少在单声道录音的噪音液量保持在较低水平，所以，它只是涵盖切片。 <ol><li>在细胞贴附配置GABA激活渠道。当您已经获得一个GΩ的密封膜在细胞贴附模式，并且可以从吸管封闭膜补丁的渠道记录（见图1B）。作为氨基丁酸不穿过细胞膜，GABA是投入液来激活补丁渠道。突触外GABA激活通道激活的延迟几分钟。该修补程序的潜力= - 吸管潜力在PClamp即当您设置的潜力 - 40 mV的，它是由膜去极化40 mV的记录。 记录在细胞贴附模式的优点是该通道是在其原生环境，并与正常细胞内的蛋白质和因素。缺点是，你不知道您正在录制的潜力，因为你不知道整个补丁的绝对静止膜电位和潜在吸管潜力和细胞的静息膜电位决定。 </li><li> GABA激活通道内而外的配置。当在细胞贴附模式下轻轻抬起你的吸管和移动使用的micromanipulators精细动作，从片方和离。现在，你有内而外的补丁，并可以记录在由内向外的模式。 您可以记录从膜的修补程序是由您的吸管封闭的（见图1B）。在细胞贴附模式您有液GABA激活的渠道。但是，脂质双层交叉，如苯二氮卓类，巴比妥类，异丙酚等药物，可以被应用在洗澡的解决方案，因为他们将能够跨到吸管和调节的渠道[14]。该补丁潜力= - 吸管的潜力，并覆盖在细胞贴附模式，因为没有静息膜电位，修补潜力等于移液器的潜力，例如，如果你设置了在PClamp控股潜力-40毫伏的补丁潜力正是40毫伏。 内配置的优点是可以应用药物或酶的内部膜表面，并研究是否影响通道的特性。内而外的补丁的力量并不适用于形成这种剽窃的补丁配置可能更渠道复杂的温柔外的补丁时形成的通道的膜环境（请参见下文） 。 </li><li> GABA激活渠道外配置。当在全细胞模式下轻轻抬起或拉回吸管，用显微精细动作。同时，您的计算机上观看的窗口，显示的补丁属性。当您绘制吸管离的细胞，你会看到电容瞬态消失，你将再次有GΩ的印章。您现在已经形成了外部的补丁，并可以记录在外部输出模式。 不知怎的，在吸管的尖端张开密封细胞膜，膜内部面临吸管内部和面临的录音室（图1B）原膜外。现在要检查药​​物GABA和其他溶解液。补丁潜力=吸管的潜力，就像在全细胞模式，例如，如果你设置PClamp持有潜力至40 mV的补丁潜力正是40毫伏。 外模式的优点是，你知道正是你的膜电位和膜原​​来的外表面的试验室，要使用的药物可以溶解液，并方便地应用于洗掉渠道。缺点是，你不再可能有连接通道的细胞内，甚至跨膜蛋白，可能需要全通道功能的因素作为密封膜和一些分子可能已丢失或当地的膜环境扭曲的过程。但是，不同的补丁配置允许在分子水平上，不与任何其他方法可能今天清扫通道属性。 </li></ol> 7。分析结果: 单通道和全细胞电流的分析与PClamp软件。我们使用Synaptosoft（GA，USA）或AXOGraph（澳大利亚悉尼，）的突触事件分析。 8。代表性的成果: 图2A显示单通道电流激活20纳米的GABA浓度，在齿状回颗粒神经元细胞贴附补丁。激活通道的最大电导的潜伏期为2分38秒和 15 PS最大电导44 。这个补丁是40 mV的去极化。图2B显示identifiying补品和阶段性电流大鼠海马神经元的全细胞电压钳当前记录的例子。 GABAA受体拮抗剂SR95531抑制的突触外（补品）和突触生成的（阶段性）GABA激活电流。我们应用（BA）齿状回神经元及（bb）胰岛素治疗的揭示补虚电流基准电流阻断突触外渠道（BA，B）和诱导SR95531转变的CA1区锥体神经元GABAA受体拮抗剂SR95531（100微米）突触电流消失时SR95531块突触通道（BB）。在基础状态下，CA1区锥体神经元不补品GABA激活电流，但是当神经元暴露生理浓度的胰岛素4 GABA激活进补电流发展。 <img src="/files/ftp_upload/2858/2858fig1.jpg" alt="图1"/> 图1A。大鼠海马脑片，地区确定CA1和CA3区锥体细胞层和齿状回（DG）的颗粒细胞层。示意图膜片钳配置。 <img src="/files/ftp_upload/2858/2858fig2.jpg" alt="图2"/> 图2A。单通道20纳米γ-氨基丁酸在大鼠海马脑片齿状回颗粒细胞在细胞贴附补丁激活电流。保持电位为40 mV的去极化（吸管潜力-40毫伏）。星号（*第一，当前的跟踪）标识的电流从那里更快的时间尺度（2所示ND电流跟踪）。A.齿状回颗粒细胞在保持电位-60 mV的电压钳模式 B. CA1区锥体神经元在大鼠海马脑片全细胞记录电流。 CA1区的实验，在室温下2小时孵育切片均录得电流之前在1纳米胰岛素。

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W., Sieghart, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+Union+of+Pharmacology.+LXX.+Subtypes+of+gamma-aminobutyric+acid(A)+receptors:+classification+on+the+basis+of+subunit+composition,+pharmacology,+and+function.">International Union of Pharmacology. LXX. Subtypes of gamma-aminobutyric acid(A) receptors: classification on the basis of subunit composition, pharmacology, and function.</a> Update. Pharmacol Rev. 60, 243-260 (2008).</li><li>Hamill, O. P., Marty, A., Neher, E., Sakmann, B., Sigworth, F. 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P., Birnir, B., Gage, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+GABA(A)+channel+conductance+increased+by+diazepam.">Hippocampal GABA(A) channel conductance increased by diazepam.</a> Nature. 388, 71-75 (1997).</li><li>Lindquist, C. E., Birnir, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graded+response+to+GABA+by+native+extrasynaptic+GABA+receptors.">Graded response to GABA by native extrasynaptic GABA receptors.</a> J Neurochem. 97, 1349-1356 (2006).</li><li>Birnir, B., Eghbali, M., Cox, G. B., Gage, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+concentration+sets+the+conductance+of+delayed+GABAA+channels+in+outside-out+patches+from+rat+hippocampal+neurons.">GABA concentration sets the conductance of delayed GABAA channels in outside-out patches from rat hippocampal neurons.</a> J Membr Biol. 181, 171-183 (2001).</li><li>Birnir, B., Eghbali, M., Everitt, A. B., Gage, P. W. B. i. c. u. c. u. l. l. i. n. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pentobarbital+and+diazepam+modulate+spontaneous+GABA(A)+channels+in+rat+hippocampal+neurons.">pentobarbital and diazepam modulate spontaneous GABA(A) channels in rat hippocampal neurons.</a> Br J Pharmacol. 131, 695-704 (2000).</li><li>Birnir, B., Everitt, A. B., Gage, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+GABAA+channels+in+rat+dentate+gyrus.">Characteristics of GABAA channels in rat dentate gyrus.</a> J Membr Biol. 142, 93-102 (1994).</li></ol>

全细胞电流的代表乐团的活动通道，在细胞内产生的电流。具体的药理学和氯化物的GABAA渠道的渗透性，使得它可以找出与这些渠道相关的电流。短暂性突触通道激活和活化突触外渠道的滋补允许GABAA受体在神经元表达的渠道，这两个种群之间容易分化。然而，只有单声道录音，可以研究通道分子的动力学性质，产生的补药电流。 100微秒内激活突触的渠道不同，进补的GABAA通道被激活GABA是适用于 4,7,15,16,17,18的时间延迟在分钟不等。这在许多通道属性的渠道，结果延迟激活不能使用全细胞记录研究。在单通道的水平，GABAA渠道的不同亚群的基础上，不同的功能和药理特性，可以识别。因此，采取什么不同的膜片钳配置所提供的优势，它可以详细研究的分子的功能和GABA激活通道在神经元产生的滋补和突触电流的药理学。

几个膜片钳硬件和软件系统，它们都使用相同的基本原则。在我们的实验室中，我们使用Axoclamp放大器和PClamp软件（Molecular Devices公司，美国），记录和分析我们的实验。另一种流行的品牌是HEKA放大器和软件（HEKA Elektronik公司，德国） Microforge:Narishige MF - 830（日本）; Vibrotome:如徕卡VT1200S（德国）或坎普登仪器vibrotome（英国）;显微镜:尼康Eclipse的E600FN;水浴; Micromanipulators:定制和Narishige micromanipulators（日本）;拖轮:DMZ通用（德国）或Narishige PC - 10（日本）。电极的玻璃:GC150F - 15（美国哈佛大学器械，英国）。

gaba-activated single-channel and tonic currents in rat brain slices

GABA的一个渠道是在目前所有的神经元都位于突触和突触外，它们产生阶段性和补品的电流，分别为4,5,6,7的 GABA A通道是一个五聚体的GABA门控的氯通道。通道亚单位被分成8个家庭（α1- 6，β1- 3，γ1- 3，δ，ε，θ，π和ρ）。两个的阿尔法，两个贝塔和一个3路亚基形成功能通道 8 。通过结合分型的具体GABA激活单通道与包括所有人群中的神经元的GABA A通道的研究分子研究成为可能，以了解抑制神经元的基本机制，以及它是如何调制药理代理。 我们利用膜片钳技术，9,10，以研究在海马脑片活着神经元的GABA A通道的功能特性，并记录单通道和全细胞电流。我们进一步研究如何GABA的浓度不同，其他药物和内和外的因素影响的渠道。如需详细的理论和实践的膜片钳方法的描述，请参阅单声道录音编辑Sakman由B和E内尔10。

Watch this Scientific Journal Video about GABA-activated Single-channel and Tonic Currents in Rat Brain Slices at JoVE.com

GABA-activated Single-channel and Tonic Currents in Rat Brain Slices

我们用膜片钳技术来衡量GABA激活单通道电流（GABAA受体通道，GABAA受体）和他们在神经元产生的突触和补品电流。激活的途径在健康和疾病的神经元的兴奋性降低<sup&gt; 1,2,3,4</sup&gt;。

在大鼠脑片的单通道和补品GABA激活电流

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, ...

gaba-activated-single-channel-and-tonic-currents-in-rat-brain-slices

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

25.1K 次观看.  Uppsala University, Sweden. 我们用膜片钳技术来衡量GABA激活单通道电流（GABAA受体通道，GABAA受体）和他们在神经元产生的突触和补品电流。激活的途径在健康和疾病的神经元的兴奋性降低 1,2,3,4。

视频: GABA-activated Single-channel and Tonic Currents in Rat Brain Slices

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 ...

科研

神经科学

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

在脑片恐惧电路体外光遗传学解剖

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

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 ...

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 ...

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 ...

Whole-cell Currents Induced by Puff Application of GABA in Brain Slices

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug application during electrophysiological recording is the puff technique, which can be used to study the effect of pharmacological reagents on neuronal activities in brain slices. The greatest advantage of puff application is that the drug concentration around the recording site increases rapidly, thus preventing desensitization of membrane receptors. Successful use of puff application involves careful attention to the following elements: the concentration of the drug, the parameters of the puff micropipette, the distance between the tip of a puff micropipette and the neuron recorded, and the duration and pressure driving the puff (pounds per square inch, psi). This article describes a step-by-step procedure for recording whole-cell currents induced by puffing gamma-aminobutyric acid (GABA) onto a neuron of a prefrontal cortical slice. Notably, the same procedure can be applied with minor modifications to other brain areas such as the hippocampus and the striatum, and to different preparations, such as cell cultures.

We describe the puff technique, by which pharmacological reagents can be administered during whole-cell patch-clamp recording, and highlight various aspects of the features that are crucial for its success.

粉扑诱导的全细胞电流在脑切片中的应用

Pharmacological administration is commonly used when conducting whole-cell patch-clamp recording in brain slices. One of the best methods of drug ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

脑切片神经元树突的双光子钙成像

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

利用体外大鼠脑切片研究变性主要事件的一种替代方法

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

鼠脑切片偶联微电极阵列癫痫活性的记录与调控

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

成人脑切片长期抑郁症诱导评估

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...