作者要感谢伊沃尔特为她出色的技术援助。由Drs生成VGAT金星转基因大鼠。华柳川，米平林和Y川口在全国生理学研究所，日本冈崎，利用博士答:宫胁提供PCS2金星。

道德声明:所有程序与动物维修均按照制度执行指引，德国动物福利法案，欧盟理事会86/609 / EEC号指令对动物的保护，地方当局（柏林，T 0215/11准则） 1，制备急性，海马<ol><li>以转基因大鼠（17〜24日龄），在VGAT子，其中标签的大部分皮质抑制性13表达的荧光金星/ YFP的蛋白质。杀头的老鼠。迅速解剖大脑（&lt;40秒）进semifrozen，carbogenated（95％O 2/5％CO 2）的基于蔗糖的人工脑脊髓液（蔗糖-ACSF， 图1A）。 </li><li>评估解剖大鼠脑金星/ YFP的荧光505 nm的LED灯和515排放过滤器，安装在一对护目镜。 </li><li>删除皮质和cereb的正面第三ellum;然后分离出半球，全部用手术刀。除去皮层的背侧表面，以提供一个平坦的表面粘上脑下，如先前所描述的14。 </li><li>切断海马结构的横切片（300微米）上vibratome，半球应semifrozen包围，carbogenated蔗糖学联（ 图1B）14。去除皮质延髓，中脑和脑干的其他地区。每片转移到含有蔗糖，学联，这是carbogenated，并加热到35ºC淹没保持室。 </li><li>离开片恢复在35°C下进入回暖学联的最后一个片段的时间为30分钟。为了激活代谢过程，并促进神经切断过程的再密封做到这一点。然后转移到室温下储存（ 图1C）。 </li></ol> 2，制作与录音移液器的灌装<ol><li>浦从玻璃毛细管LL补丁移液管，以便为2-4MΩ移液管电阻时填充有过滤实现（注射器过滤器，孔径:0.2微米），含有0.1％生物胞素（用于胞内标记）的细胞内溶液。请在冰上冷却，以防止其成分的降解细胞内液。 </li><li>填写补丁移液器识别性突触后电流与含有生理相关的低氯溶液-浓度（E R（氯离子）= -61毫伏;看到解决方案的列表）。 </li><li>用于配对的记录，以确定在突触前受体介导的反应，填充修补移液器与低的 Ca 2 +的缓冲能力，以防止与发射机释放干扰突触前，以及4倍高氯细胞内液-浓度（E R（氯离子）= -20毫伏），以改善信号对噪声观测IPSC的5允许药理RESP的准确评估的onsiveness。需要注意的是不断变化的Cl -的浓度可以改变IPSC动力学15。 </li></ol>从FS-INS 3全细胞膜片钳记录<ol><li> Carbogenate的ACSF和通过灌注系统馈送到记录室中，用蠕动泵（它也消除ACSF从录音室经由吸入管线， 图2A）的装置。在准备打开在设置中的所有设备进行记录。 </li><li>转片到录音室，并固定在其位置与铂金戒指串成单纤维丝。定位片，使CA1区的地层（STR）锥体垂直贯穿的视野，允许访问与2移液器两个海峡。 radiatum和STR。 oriens同时（ 图2C和图4A）。 </li><li>将腔进入设置和启动carbogenated灌注和加热（32-34℃）录制的相关CSF为5-10毫升/分钟的流速。 </li><li>评估下的IR-DIC的光学切片质量的40倍物镜放大倍数，并用可视化的CCD摄像头在显示器上观看。设好片的质量，如果有大量圆形，中等对比CA1区锥体细胞（CA1电脑）都可以在海峡可见。锥体深度为20〜30微米以下的顺利，轻轻陨石坑的表面（ 图2C）。质量差的片含有大量的高对比度，萎缩或肿胀的细胞，具有不均匀的片表面。 </li><li>下表荧光照明识别推定的FS的interneurons那些表达金星/ YFP（ 图2B），并在或靠近STR大的多极胞体。锥体 。相当深，以切片（50-100微米， 图2C）中选择单元格，以便更好地维护自己的形态完整。 </li><li>安装在上的探头的移液管固定器的记录电极;然后应用程序立法院通过管线低，正压力（20-30毫巴）。降低吸移管向片的表面上，稍有偏移到所选择的神经元的中心。 </li><li>获得全细胞记录的配置如前所述14,16和还可参见图2D和2E: <ol><li>靶细胞:增大压力，以70-80毫巴，并通过切片所选择的小区（ 图2D中 ，上部）的体细胞向正上方迅速降低吸移管。 </li><li>接近电池:按吸管对细胞膜产生一个"酒窝"就可以了（ 图2D，上图）。迅速地执行该步骤，以防止相邻小区中的生物胞素标记。 </li><li>创建一个千兆欧姆封:释放压力，并同时应用20 mV的电压指令的吸管。 Ä千兆欧姆封（1-50GΩ; 图2D，底部和图2E中）炎热的典型盟友迅速发展。一旦密封，申请预期静息膜电位（通常介于-70和-60毫伏）作为电压指令。 </li><li>突破的补丁:一旦密封，破裂的膜修补用的负压的短脉冲;由此实现了全细胞构型（ 图2E，底部）。 </li></ol></li><li>补偿的全细胞电容和串联电阻（R S）。 R 5 为通常5-20MΩ，稳定长达120分钟。抛弃细胞，如果膜电位（V M）上的突破比-50毫伏以上的去极化; R 5是比最初的30MΩ更大;或R S发生变化超过20％以上的记录的过程。 </li><li>确定FS-INS通过他们的反应（在电流钳模式），以一个家庭hyper-对去极化电流脉冲（-250〜250 pA的， 图2F，上图）。 FS-INS已经相对去极化V M（通常是-50吨Ø-60毫伏），短膜时间常数（&lt;20毫秒），并响应500 pA的去极化电流注入与动作电位火车（APS）在频率&gt; 100 Hz的11（ 图2F，下图），这是明显的有别于海马CA1电脑（ 图2F，中）。 </li></ol> 4，外电刺激唤起的GABA 乙 R-介导的反应<ol><li>在海峡的边界切片:;观察突触诱发反应，位置的细胞外刺激电极（0.1〜0.3MΩ电阻贴片吸管充斥着2 M氯化钠）。 radiatum和STR。腔隙-moleculare。放置电极200-300微米横向于体细胞，防止细胞的直接电刺激，并尽量减少刺激伪影（ 图3A）。 </li><li>一旦刺激电极被定位，得到的全细胞记录所选择的细胞，并评估生理学表型在电流钳模式，如第3.9节（ 图3B）。 </li><li>记录在电压钳（V M -65毫伏）的神经元，突触传递轴突的电刺激，在50 V（〜500μA有效的刺激），每20秒，使用独立的恒压刺激。使用单一刺激（100微秒的持续时间， 如图3C所示 ，顶端）观察GABA; B R介导的IPSC的，并具有多种刺激的列车交错（在200赫兹），以产生更大的发送器释放。 </li><li>巴斯申请离子型谷氨酸受体拮抗剂（AMPA受体:DNQX [10 M]。NMDA受体:D-AP5 [50μm]的）揭示孤立突触IPSC（ 图3C，中上部）。进一步孤立氨基丁酸乙 R-介导的IPSC与应用程序的GABA A R阻滞剂（gabazine [10 M]。 图3C中L奥尔）。 </li><li>确认所产生慢外向钾电流（ 图3C低，扩）为被氨基丁酸乙的R-介导的后续应用的CGP-55845 [5微米]（ 图3C较低，伏在灰色） </li></ol>突触的5成对录音加上FS-IN和CA1电脑<ol><li>评估抑制性突触传递的具有同步记录，如下所述突触耦合器IN和PC机对之间进行的GABA 乙 R-介导的突触前的控制。 </li><li>首先，建立突触前interneuron的全细胞记录（如在第3节），并确认FS型（ 图4A）。 </li><li>然后修补邻近CA1电脑（20-100微米的距离， 图4A），并应用简单阈上的去极化电流脉冲（1毫秒的时间，1-5 nA的幅度）的突触前输入（电流钳模式持有）引出的AP。如果突触合作nnection存在时，在IN结果在CA1的PC，在电压钳保持IPSC的接入点（补偿R 5至约80％）。 </li><li>如果有必要，填补进一步CA1区个人电脑的新记录电极和记录，直至找到一个连接。 </li><li>一旦建立了连接，引起对受影响的突触前的FS-IN来评估两者的单一的突触响应和动态行为。使用2去极化刺激典型的双脉冲协议与50毫秒的间隔（ 图4B）。 </li><li>收集在基准条件控制的痕迹。然后，应用选择性的GABA; B R激动剂巴氯芬（10μM）的灌流ACSF，从而激活GABA 乙卢比，其次是拮抗剂CGP-55845（5μM），以完全阻止该受体介导的作用。在对每种药物的条件（ 图4B和C）稳态收集〜50的痕迹。 </li><li>一旦录音完成后，通过形成outsid密封体膜电子出补丁:慢慢从细胞体抽出吸管在V形夹紧和作为R 5的增加，降低了V M至-40毫伏。这样做是为了便于外出补丁的形成;然后从浴吸管。 </li></ol>电生理特性的分析6。 <ol><li>注:多种不同的软件包可用于收购电数据。在这里，WinWCP，在免费的斯特拉斯克莱德电软件包Windows程序时，它可录制高达16个模拟输入通道和10个数字信号输出。 </li><li>低通滤波器的所有数据，在5〜10 kHz和样品在20千赫。 </li><li>与离线分析套件分析生理数据。 注:Stimfit，一个开源的软件包，其中包括一个Python的外壳，用在这种情况下;但是其他方法可以很容易地被使用。 <ol><li>分析被动膜糖蛋白记录神经元的roperties，在电流钳收购，从静止膜电位。 </li><li>测量从记录开始记录响应的基线的平均静息膜电位。 </li><li>计算输入电阻，用欧姆定律，从电压响应于最小的超极化电流脉冲（≤-50 PA）。为了改善信噪比，平均的多次波轨迹。注:我们的例子是典型的10-50个人的扫描平均值。 </li></ol></li><li>估计表观膜时通过安装一个单指数曲线的响应最小的超极化电流脉冲的衰减时间常数。 </li><li>分析动作电位波形来确定的阈值，振幅（阈值到峰值）和持续时间（宽度的一半高度测量）根据阈值水平去极化电流脉冲引起。 </li><li>分析GABA; B R的电压钳记录介导的IPSC的。滤波器跟踪离线处500赫兹（高斯滤波器），并评估该峰振幅和潜伏期的GABA; B R介导的应答的（在至少10痕迹的平均值）。 </li><li>检测γ -氨基丁酸乙卢比对土著民族的抑制输出的峰值和前面的基线之间测得的GABA A R-介导的IPSC的峰值振幅变化的影响。从≥50痕迹的控制周期和所有药物时代的稳态计算的平均幅度。 </li></ol> 7，可视化及免疫细胞化学FS宏<ol><li>以下的录音，通过浸渍固定切片在4％多聚甲醛的0.1M磷酸盐缓冲液（PB，pH值= 7.35），O 2 / N，4℃。 </li><li>如果必要的切片可以处理之前被转移到PB和存储长达〜1周时间。 </li><li>洗涤切片用大量新鲜的PB，并随后在0.025M的PB用0.9％的NaCl（PBS，pH值= 7.35）。 </li><li>为了减少非特异性抗体结合，阻断FO切片中，R 1小时，在室温在含有10％正常山羊血清（NGS），0.3％的Triton-X100（清洁剂通透膜）和0.05％NaN 3的，由在PBS中的溶液。 </li><li>标记为光伏表达，使用抗光伏单克隆小鼠抗体稀释在含有5％NGS，0.3％的Triton-X100，0.05％NaN 3的，在PBS中的溶液。在4℃下孵育12的初级抗体为2-3天。冲洗切片彻底的PBS中。 </li><li>应用荧光抗小鼠的二抗（ 例如，Alexafluor-546。）连同生物素结合蛋白链菌素，缀合至荧光染料（ 例如 ，Alexafluor-647）;并培养在含3％农工商，0.1％的Triton-X100，0.05％NaN 3的解决方案，用PBS稀释，并培育在o / n 4℃。 </li><li>宽松冲洗2-3倍，用PBS其次是2-3中的冲洗PB片。装在载玻片上的切片。使用300微米琼脂垫片，以防止片坍塌。盖滑片与fluores分装中，密封，钉清漆。 </li></ol> 8，成像和可视化重建FS宏<ol><li>可视化利用共聚焦显微镜切片，与fluorochome记者兴奋与适当的激光线（激光二极管635 nm处Alexafluor-647;氦氖543 nm处Alexafluor-546标签的PV和氩气488或515纳米的金星/ YFP）。 </li><li>拍摄图像在适当的Z分辨率（通常为0.5-1微米的步骤，用20倍的目标）产生一个Z堆栈整个细胞。多个堆栈通常需要将图像的整个细胞，其可以被数字缝合离线使用斐济/ ImageJ的软件（ 图5A）。 </li><li>用半自动跟踪的方法重建的拼接图像的堆栈单元（简单轴突示踪插件斐济/ ImageJ的软件包17， 图C）。 </li><li>最后，评估光伏免疫反应的Interneuron具有高数值孔径的物镜（60X硅浸渍，NA = 1.3）。使胞体的图像，近端树突和轴突近端，或者对轴突末梢，如果光伏发电体冲刷太强。细胞被认为是阳性的光伏如果免疫标记看出，以符合生物胞素标记的结构（ 图5B）。 </li></ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+GABAB-Receptor-Mediated+Effects+in+Perisomatic-+and+Dendrite-Targeting+Parvalbumin+Interneurons.">Differential GABAB-Receptor-Mediated Effects in Perisomatic- and Dendrite-Targeting Parvalbumin Interneurons.</a> Journal of Neuroscience. 33 (18), 7961-7974 (2013).</li><li>Uematsu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+chemical+composition+of+cortical+GABAergic+neurons+revealed+in+transgenic+Venus-expressing+rats.">Quantitative chemical composition of cortical GABAergic neurons revealed in transgenic Venus-expressing rats.</a> Cerebral Cortex. 18, 315-330 (2008).</li><li>Bischofberger, J., Engel, D., Li, L., Geiger, J. R. P., Jonas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch-clamp+recording+from+mossy+fiber+terminals+in+hippocampal+slices.">Patch-clamp recording from mossy fiber terminals in hippocampal slices.</a> Nature Protocols. 1, 2075-2081 (2006).</li><li>Houston, C. M., Bright, D. P., Sivilotti, L. G., Beato, M., Smart, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Chloride+Ions+Regulate+the+Time+Course+of+GABA-Mediated+Inhibitory+Synaptic+Transmission.">Intracellular Chloride Ions Regulate the Time Course of GABA-Mediated Inhibitory Synaptic Transmission.</a> Journal of Neuroscience. 29 (33), 10416-10423 (2009).</li><li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+techniques+for+studying+ionic+channels+in+excitable+membranes.">Patch clamp techniques for studying ionic channels in excitable membranes.</a> Annual Review Physiology. 46, 455-472 (1984).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji+an+open-source+platform+for+biological-image+analysis.">Fiji an open-source platform for biological-image analysis.</a> Nature Methods. 9, 676-682 (2012).</li><li>Geiger, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch-clamp+recording+in+brain+slices+with+improved+slicer+technology.">Patch-clamp recording in brain slices with improved slicer technology.</a> Pflugers Archives. 443, 491-501 (2002).</li><li>Malinow, R., Tsien, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+enhancement+shown+by+whole-cell+recordings+of+long-term+potentiation+in+hippocampal+slices.">Presynaptic enhancement shown by whole-cell recordings of long-term potentiation in hippocampal slices.</a> Nature. 346, 177-180 (1990).</li><li>Vida, I., Bartos, M., Jonas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shunting+inhibition+improves+robustness+of+gamma+oscillations+in+hippocampal+interneuron+networks+by+homogenizing+firing+rates.">Shunting inhibition improves robustness of gamma oscillations in hippocampal interneuron networks by homogenizing firing rates.</a> Neuron. 49 (1), 107-117 (2006).</li><li>Neu, A., F&#246;ldy, C., Soltesz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postsynaptic+origin+of+CB1-dependent+tonic+inhibition+of+GABA+release+at+cholecystokinin-positive+basket+cell+to+pyramidal+cell+synapses+in+the+CA1+region+of+the+rat+hippocampus.">Postsynaptic origin of CB1-dependent tonic inhibition of GABA release at cholecystokinin-positive basket cell to pyramidal cell synapses in the CA1 region of the rat hippocampus.</a> Journal of Physiology. 578 (1), 233-247 (2007).</li><li>Baude, A., Bleasdale, C., Dalezios, Y., Somogyi, P., Klausberger, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoreactivity+for+the+GABAA+receptor+alpha1+subunit,+somatostatin+and+Connexin36+distinguishes+axoaxonic,+basket,+and+bistratified+interneurons+of+the+rat+hippocampus.">Immunoreactivity for the GABAA receptor alpha1 subunit, somatostatin and Connexin36 distinguishes axoaxonic, basket, and bistratified interneurons of the rat hippocampus.</a> Cerebral Cortex. 17, 2094-2107 (2007).</li></ol>

作者宣称，他们没有竞争的经济利益。

我们形容它结合了电生理和神经解剖技术在体外功能鉴定morphologically-和确定的neurochemically神经元的方法;尤其是皮质的抑制诸IN的不同类型。该过程的主要方面是:（1）预选择潜在诸IN的; （2）细胞内记录和神经元的可视化;最后（3）记录的土著民族形态学及免疫细胞化学分析。虽然本研究中特别讨论的PV-INS，所描述的协议，可用于从任何interneuron或其他神经元的类型，以最少的改动类似录像。 <p...

海马神经元电路一直是严格审查的主体，对于两者的解剖和生理，由于人类和啮齿类动物学习和记忆的重要作用以及空间导航。同样，海马突出，但简单的分层组织，使本地区的研究，解决皮层网络的结构和功能特性青睐的主题。 海马电路是由兴奋性主细胞（&gt; 80％）和一个较小的（10-20％），但抑制性1-3的高度多样化的队列。释放的interneurons从他们的轴突末梢，其作用在快离子型GABA受体γ-氨基丁酸（GABA）（GABA A RS）和慢代谢型GABA受体乙 （GABA 乙 RS）4。这些抑制机制抵消激励和调节主细胞的兴奋性，从而IR时间和放电模式。然而，从GABA释放的interneurons行为不仅对主细胞，同时也对自己的interneurons 5,6。前和突触后受体介导的各类interneuron之间的反馈调节和抑制的相互交互。在interneuron网络这些抑制机制被认为是中心的产生和人口活动模式成形，特别是振动在不同的频率7。 全细胞膜片钳记录是一种行之有效的方法固有特性和神经元的突触相互作用的检查。然而，由于interneuron类型的高多样性，抑制性的调查，需要严格识别所记录的细胞。作为海马interneuron类型的特点是鲜明的形态特征和神经化学标志物的表达，结合解剖和免疫Ëxamination可以提供一种方法来确定精确的interneuron身份6,8,9。 在本论文中，我们描述了一种实验方法，其中从单个神经元或突触耦合对全细胞膜片钳记录是结合胞内标记，随后事后形态学和免疫细胞化学分析，允许缓慢GABA B中的特征受体介导的中鉴定的interneurons抑制作用。作为一个例子，我们专注于interneuron的一个主要类型，即所谓的"篮状细胞"（BC）的一个子集，它支配其突触后目标的胞体和树突的近端和的特点是"快扣球"（FS）放电模式中的钙结合蛋白小清蛋白（PV）10,11，一个轴突密集覆盖的电池主体层，并表达。这些显示的interneurons大的抑制性突触后电流，以及突出的前它们的突触输出突触调节，以应对GABA; B R激活12。这里所描述的技术的组合可用于同样，调查在各种其他确定神经类型的本征或突触机制。

<table><tbody><tr><td>Transgenic vGAT-venus rats</td><td></td><td></td><td>see Uematsu , &lt;em&gt;et al.&lt;/em&gt;, 2008</td></tr><tr><td>Venus (515 nm) goggles</td><td>BLS Ltd., Hungary</td><td></td><td></td></tr><tr><td>Dissection tools</td><td>&lt;em&gt;i.e.&lt;/em&gt; FST</td><td></td><td>For brain removal</td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1200S</td><td>Or other high end vibratome with minimal vertical oscillation</td></tr><tr><td>Slice holding chambers</td><td></td><td></td><td>Custom-made in lab</td></tr><tr><td>Upright IR-DIC microscope</td><td>Olympus, Japan</td><td>BX50WI</td><td>With micromanipulator system; &lt;em&gt;i.e.&lt;/em&gt; Luigs and Neumann, Kleindiek &lt;em&gt;etc.&lt;/em&gt;</td></tr><tr><td>CCD camera</td><td>Till Photonics</td><td>VX55</td><td></td></tr><tr><td>505 nm LED system</td><td>Cairn Research</td><td>OptiLED system</td><td>Or mercury lamp or other LED system &lt;em&gt;i.e.&lt;/em&gt; CooLED.&amp;nbsp;</td></tr><tr><td>Multiclamp 700B&amp;nbsp;</td><td>Axon Instruments</td><td></td><td>Alternatively 2x Axopatch 200B amplifiers&amp;nbsp;</td></tr><tr><td>WinWCP acquisition software</td><td>John Dempster, Strathclyde University</td><td></td><td>Any quality acquisition software could be used, &lt;em&gt;i.e.&lt;/em&gt; EPHUS, pClamp, Igor etc.&amp;nbsp;</td></tr><tr><td>Electrode Puller</td><td>Sutter</td><td>P-97</td><td>Used with box-filament</td></tr><tr><td>Borosilicate pipette glass</td><td>Hilgenberg, Germany</td><td>1405020</td><td>2 mm outer, 1 mm inner diameter, no filament</td></tr><tr><td>Peristaltic pump</td><td>Gilson</td><td>Minipuls</td><td>Other pumps or gravity feed could be used instead</td></tr><tr><td>Digital Thermometer</td><td></td><td></td><td>Custom made</td></tr><tr><td>Digital Manometer</td><td>Supertech, Hungary</td><td></td><td></td></tr><tr><td>&lt;a href=&quot;http://digitimer.com/products/research/stimulators/&quot; target=&quot;_blank&quot;&gt;Isolated constant voltage stimulator&lt;/a&gt;</td><td><a href="http://www.digitimer.com" target="_blank"><img src="/img/new_window.png" /> Digitimer Ltd.</a></td><td>&lt;a href=&quot;http://digitimer.com/products/research/stimulators/ds2a-isolated-constant-voltage-stimulatorstimulus-isolator/&quot; target=&quot;_blank&quot;&gt;DS-2A&lt;/a&gt;</td><td></td></tr><tr><td>Biocytin</td><td>Invitrogen</td><td>B1592</td><td>Otherwise known as &amp;epsilon;-Biotinoyl-L-Lysine&amp;nbsp;</td></tr><tr><td>DL-AP5(V) disodium salt</td><td>Abcam Biochemicals</td><td>ab120271</td><td></td></tr><tr><td>DNQX disodium salt</td><td>Abcam Biochemicals</td><td>ab120169</td><td>Alternatively NBQX or CNQX</td></tr><tr><td>Gabazine (SR95531)</td><td>Abcam Biochemicals</td><td>ab120042</td><td>Alternatively bicuculline methiodide</td></tr><tr><td>R-Baclofen</td><td>Abcam Biochemicals</td><td>ab120325</td><td></td></tr><tr><td>CGP-55,845 hydrochloride</td><td>Tocris</td><td>1248</td><td></td></tr><tr><td>Streptavidin 647</td><td>Invitrogen</td><td>S32357</td><td></td></tr><tr><td>anti-PV mouse monoclonal antibody</td><td>Swant, Switzerland</td><td>235</td><td>Working concentration 1:5,000-1:10,000</td></tr><tr><td>anti-mouse secondary antibody&amp;nbsp;</td><td>Invitrogen</td><td>A11030</td><td>If using Venus or GFP rodent using a red-channel (&lt;em&gt;i.e.&lt;/em&gt; 546 nm) is advisable.</td></tr><tr><td>Normal Goat Serum</td><td>Vector Labs</td><td>S-1000</td><td></td></tr><tr><td>Microscopy slides</td><td></td><td></td><td>Any high quality brand</td></tr><tr><td>Glass coverslips</td><td></td><td></td><td>Usually 22 x 22 mm</td></tr><tr><td>Agar spacers</td><td></td><td></td><td>Agar block, cut on vibratome to 300 &amp;mu;m</td></tr><tr><td>Laser scanning confocal microscope</td><td>Olympus, Japan</td><td>Fluoview FV1000</td><td>Or other comparable system</td></tr><tr><td>Fiji (Fiji is just ImageJ)</td><td>http://fiji.sc/Fiji</td><td></td><td>See Schindelin &lt;em&gt;et al.&lt;/em&gt;, 2012</td></tr></tbody></table>

whole-cell patch-clamp recordings from morphologically- and neurochemically-identified hippocampal interneurons

GABA能抑制性脑部神经回路中发挥核心作用。的interneurons包括神经元群（10-20％）的一小部分，但表现出的生理，形态和神经化学异质性很高的水平，反映了他们不同的功能。因此，的interneurons的调查提供了重要的见解的组织原则和神经回路的功能。然而，这需要一个综合的生理和解剖学的方法对个别interneuron类型的选择和鉴定。全细胞膜片钳从转基因动物中的急性脑切片，表达下的interneuron特异性标志物的启动子的荧光蛋白的记录，提供了一种有效的方法来定位和电生理学表征特定interneuron类型的特性和突触性质。结合细胞内的染料标记，这种方法可以扩展事后MOrphological及免疫细胞化学分析，使记录的神经元系统的鉴定。这些方法可以被定制，以满足范围广泛的方面的皮层神经元的不同类型的功能性的科学问题。

只要切片质量明显好，无论从CA1电脑和FS的插件可以用最少的困难，实现记录。在VGAT子13表示金星/ YFP的转基因鼠线不明确标识的FS-INS，或实际上的BC。然而，在和周围STR从诸IN录音。锥体 ，其中fs插件的密度通常很高1，结果在选择FS-INS（ 图2B）的概率较高。 FS的插件可以通过其特有的生理特性与两个CA1区的PC和RS-INS的不同来区分。他们有一个相对去极化?...

Watch this Scientific Journal Video about Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons at JoVE.com

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

皮层网络由一个小的，但不同组抑制性控制。因此的interneurons功能调查需要有针对性的记录和严格的鉴定。这里描述的是一种涉及从单个或突触耦合对神经元细胞内的标记，事后的形态学和免疫细胞化学分析的全细胞记录相结合的方法。

从Morphologically-和确定的Neurochemically海马的interneurons全细胞膜片钳记录

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

whole-cell-patch-clamp-recordings-from-morphologically

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

24.4K 次观看.  Charité Universitätmedizin. 皮层网络由一个小的，但不同组抑制性控制。因此的interneurons功能调查需要有针对性的记录和严格的鉴定。这里描述的是一种涉及从单个或突触耦合对神经元细胞内的标记，事后的形态学和免疫细胞化学分析的全细胞记录相结合的方法。 

视频: Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

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 ...

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 ...

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 ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

从黑质多巴胺神经元的Somatodendritic域亚细胞的膜片钳记录

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent improvements in methodologies and applications for the study of theta oscillations. A simple characterization of the isolated hippocampal preparation is presented whereby the relationship between internal hippocampal theta oscillators is examined together with the activity of pyramidal cells, and GABAergic interneurons, of the cornu ammonis-1 (CA1) and subiculum (SUB) areas. Overall, we show that the isolated hippocampus is capable of generating intrinsic theta oscillations in vitro and that rhythmicity generated within the hippocampus can be precisely manipulated by optogenetic stimulation of parvalbumin-positive (PV) interneurons. The in vitro isolated hippocampal preparation offers a unique opportunity to use simultaneous field and intracellular patch-clamp recordings from visually-identified neurons to better understand the mechanisms underlying theta rhythm generation.

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

Here, we present a protocol for recording rhythmic neuronal network theta and gamma oscillations from an isolated whole hippocampal preparation. We describe the experimental steps from extraction of the hippocampus to details of field, unitary and whole-cell patch clamp recordings as well as optogenetic pacing of the theta rhythm.

调整在海马Theta乐队<em&gt;体外</em&gt;:从孤立的啮齿动物皮下注射电路记录的方法

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent ...

One-channel Cell-attached Patch-clamp Recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

单通道细胞贴附膜片钳记录

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

生物学

Hypothalamic Kisspeptin Neurons as a Target for Whole-Cell Patch-Clamp Recordings

Kisspeptins are essential for the maturation of the hypothalamic-pituitary-gonadal (HPG) axis and fertility. Hypothalamic kisspeptin neurons located in the anteroventral periventricular nucleus and rostral periventricular nucleus, as well as the arcuate nucleus of the hypothalamus, project to gonadotrophin-releasing hormone (GnRH) neurons, among other cells. Previous studies have demonstrated that kisspeptin signaling occurs through the Kiss1 receptor (Kiss1r), ultimately exciting GnRH neuron activity. In humans and experimental animal models, kisspeptins are sufficient for inducing GnRH secretion and, consequently, luteinizing hormone (LH) and follicle stimulant hormone (FSH) release. Since kisspeptins play an essential role in reproductive functions, researchers are working to assess how the intrinsic activity of hypothalamic kisspeptin neurons contributes to reproduction-related actions and identify the primary neurotransmitters/neuromodulators capable of changing these properties. The whole-cell patch-clamp technique has become a valuable tool for investigating kisspeptin neuron activity in rodent cells. This experimental technique allows researchers to record and measure spontaneous excitatory and inhibitory ionic currents, resting membrane potential, action potential firing, and other electrophysiological properties of cell membranes. In the present study, crucial aspects of the whole-cell patch-clamp technique, known as electrophysiological measurements that define hypothalamic kisspeptin neurons, and a discussion of relevant issues about the technique, are reviewed.

Here, we present a protocol to perform a whole-cell patch-clamp on brain slices containing kisspeptin neurons, the primary modulator of gonadotrophin-releasing hormone (GnRH) cells. By adding knowledge about kisspeptin neuron activity, this electrophysiological tool has served as the basis for significant advancements in the neuroendocrinology field over the last 20 years.

下丘脑Kisspeptin神经元作为全细胞膜片钳记录的靶标

Kisspeptins are essential for the maturation of the hypothalamic-pituitary-gonadal (HPG) axis and fertility. Hypothalamic kisspeptin neurons located in ...

<meta charset="utf8"></head><body>成年小鼠海马小胶质细胞的电生理记录

Isolation and Whole-Cell Patch-Clamp Recording of Hippocampal Microglia from Adult Mice

Microglia are resident immune cells in the brain that interact with neurons to maintain the homeostasis of the central nervous system (CNS). Studies show that the microglial surface expresses potassium channels that regulate microglial activation, while abnormalities in these potassium channels can lead to neural diseases. Currently, whole-cell patch-clamp recordings of microglia are mostly performed on cultured primary microglia from fetal or newborn mice due to difficulties in conducting electrophysiological evaluations on acutely isolated microglia. This study introduces an easy-to-follow protocol for isolating hippocampal microglia from adult mice and performing whole-cell patch-clamp recordings on the isolated cells. Briefly, the brain was removed from a mouse after decapitation, the hippocampus was dissected bilaterally, and microglia were isolated using an adult mouse brain dissociation kit. The microglia were then purified using a magnetic-activated cell sorting (MACS) method and seeded onto coverslips. Successful microglial isolation was confirmed by immunofluorescent staining with anti-CD11 and anti-Iba1 antibodies. A cover slip was placed in a recording chamber, and the whole-cell potassium currents of the acutely isolated microglia were recorded under voltage-clamp conditions.

<meta charset="utf8"></head><body>成年小鼠海马小胶质细胞的分离和全细胞膜片钳记录

The present protocol describes how to isolate and purify primary hippocampal microglia from adult mice, followed by instructions for conducting whole-cell patch-clamp recordings on these acutely isolated cells.

成年小鼠海马小胶质细胞的分离和全细胞膜片钳记录

Microglia are resident immune cells in the brain that interact with neurons to maintain the homeostasis of the central nervous system (CNS). Studies show ...

从Morphologically-和确定的Neurochemically海马的interneurons全细胞膜片钳记录

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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

在器官型海马脑片搭配全细胞记录

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

在脑片全细胞膜片钳记录

从黑质多巴胺神经元的Somatodendritic域亚细胞的膜片钳记录

调整在海马Theta乐队

单通道细胞贴附膜片钳记录

下丘脑Kisspeptin神经元作为全细胞膜片钳记录的靶标

成年小鼠海马小胶质细胞的分离和全细胞膜片钳记录

成年小鼠海马小胶质细胞的分离和全细胞膜片钳记录