Financial support from the Flanders Research Foundation FWO (contract n. 12C9112N to DL), the 7th Framework Programme of the European Commission (Marie Curie Network &#8220;C7&#8221;, contract n. 238214; ICT Future Emerging Technology &#8220;ENLIGHTENMENT&#8221; project, contract n. 306502), the Interuniversity Attraction Poles Program initiated by the Belgian Science Policy Office (contract n. IUAP-VII/20), and the University of Antwerp is kindly acknowledged.

这里描述的协议符合的建议和安特卫普大学生物医学科学系的伦理委员会的指导方针。此协议需要从幼年大鼠的移出脑，由批准人道的安乐死技术得到的制剂的非有情材料。 1，设备的准备<ol><li>安装和配置数据采集和激励机制。 <ol><li>使用配有由喜剧作家支持的数据采集（DAQ）卡的个人计算机（PC）的记录信号和模拟控制电压发送到电放大器。 注:喜剧作家是一个Linux模块和库，支持从最常见的厂家众多的数据采集卡:参观http://www.comedi.org了解更多信息。 </li><li>万一一个计算机控制的膜片钳放大器是在使用中，采用一个第二PC机除了一专用于放大器控制。 注意:虽然后者可能运行的常规操作系统，额外的PC将通过一个特殊的操作系统的装置中的实时操作系统。在这些条件下，方便的是使用一个单一的显示器，鼠标和键盘连接于额外的PC，在连接由远程桌面应用程序的专用PC。 </li><li>下载包含一个实时Linux操作系统的LCG的Live CD的ISO映像预装从http://www.tnb.ua.ac.be/software/LCG_Live_CD.iso并刻录在一张空白CD或USB记忆棒" 。 </li><li>只需将CD插入包含数据采集卡的PC的驱动器，并启动它。另外，来自其在线源代码库运行Linux操作系统（ 例如，Debian的还是Ubuntu）的PC上安装LCG。咨询在线手册上的安装过程的详细信息。该手册可在网上​​http://danielelinaro.github.io/dynclamp/lcg_manual.pdf。 </li><li>引导从Live CD:THI旨意自动加载一个完全配置的系统。要做到这一点，将LCG的Live CD放入计算机的CD-ROM驱动器，并从CD引导计算机;选择实时内核（默认选项）只要启动菜单出现，并等待系统初始化。 </li><li>通过键入在命令提示符下校准数据采集卡: 须藤comedi_calibrate 要么 须藤comedi_soft_calibrate 根据数据采集板是否支持硬件或软件校准，分别为（使用命令sudo comedi_board_info获得在董事会的信息）。 </li><li>设置适当的模拟 - 数字和数字 - 模拟转换因子:这需要具有进入蜂窝电放大器的说明书，特别是涉及其规格上其转换系数。 </li><li>使用文本编辑器来指定适当的数值在文件/home/user/.lcg-env，对于环境变量AI_CONVERSION_FACTOR_CC，AI_CONVERSION_FACTOR_VC，AO_CONVERSION_FACTOR_CC，AO_CONVERSION_FACTOR_VC。 注意:这些代表输入（AI）和输出（AO）增益电流钳（CC）和电压钳（VC）的模式，并且由计算机中提供的电压指令和电流或电压之间的转换系数由放大器产生的分别。 </li><li>另外，使用提供（LCG找到的转换因子）的LCG脚本，找他或她的系统的转换系数。 注意:通过计算出的值LCG-找到转换因子是猜测，这在某些情况下需要是数字截断或舍入，以反映转换系数的精确值。 </li><li>使用LCG找到的转换因子，通过连接"细胞模型"与该放大器到相应的探头经常购买的开始。然后，打开您正在运行的Live CD，并在shell提示符下输入以下命令在Linux机器上的终端: LCG-找到转换因子-I $ HOME / .lcg-ENV -o $ HOME / .lcg-ENV 注:在这两种情况下（ 即 /home/user/.lcg-env或使用的LCG找到的转换因子手动修改），关闭和打开，以使更改生效终端。 </li><li>如果多个的探头的使用，设置转换因子所有通道中的相同的值;如果这是不可能的，请咨询在线LCG手册，以了解如何使用LCG刺激或如何将多个换算系数，以产生更好的满足用户的需求配置文件。 </li></ol></li></ol>从体感皮层2.准备急性脑片<ol><li>准备电的解决方案。 <ol><li>通过混合（以mM计）125氯化钠，2.5氯化钾，制备人工脑脊髓液（ACSF）1.25的NaH 2 PO 4，26的NaHCO 3，25葡萄糖，2的CaCl 2，和1的MgCl 2。制备10倍储备溶液，以减少准备时间上的实验当天。准备2升，其中的一个将被用于制备切片的，另一个用于记录。 </li><li>饱和用95％ 的 O 2和5％的CO 2的ACSF为之前的程序的开头至少30分钟。 </li><li>对于电流钳记录，使用含有（以mM计）的胞内溶液（ICS）115葡萄糖酸钾，20的KCl，10 HEPES，4的ATP的Mg，0.3的Na 2 -GTP，10的Na 2 -phosphocreatine。制备在冰冷溶液中，并事先将其过滤到记录的开始到消除堵塞吸管的风险。 </li></ol></li><li>大脑提取。 <ol><li>麻醉动物放置在动物感应室4％，异氟醚和快速使用断头台或大剪刀杀头的。 </li><li>削减沿中线的皮肤，其滑动至耳根。 </li><li>用细剪刀剪沿中线的头骨。保持刀片尽可能靠近到日E面，以便最小化损坏底层大脑。用镊子打开颅骨，用刮刀切断视神经和脑干，轻轻放下大脑在冰冷的学联。 </li><li>分离小脑和两个半球用手术刀（刀24）。 </li><li>从两个半球中卸下多余的水分和胶水使用一滴强力胶的倾斜平台上。迅速在脑中添加几滴ACSF的，并将其传输到vibratome室。 注意:当制备矢状切片，该平台的角度是非常重要的，以避免在切割过程期间损坏锥体细胞的树突。 </li></ol></li><li>制备切片。 <ol><li>定位在叶片上的大脑，并丢弃所述第一2.5 - 3毫米。调整的速度和频率以限制损坏片的表面，而在同一时间最小化所需的切片过程的时间。 </li><li>设置厚度为300μ米和开始切片。一旦刀片已经过去皮层，用剃​​刀刀片或弯曲的针，以切割上述海马和在感兴趣的皮层区的边缘。 </li><li>放置在一个多井孵育室的切片保持在32 - 34℃。 </li><li>缩回刀片和重复点2.3.2和2.3.3至5 - 8片切割。最好片通常是那些其中血管平行于表面。 </li><li>孵育切片30分钟后，最后一个切片被放置在腔室中。 </li></ol></li></ol>从5层锥体神经元3.膜片钳记录<ol><li>将切片录音室中和搜索健康细胞。这些细胞通常具有较低的对比度，平滑的外观和不肿大。 </li><li>检查用40X放大倍率透镜在显微镜下的切片，并搜索细胞层5中，位于约600至1000微米，从大脑的表面。 </li&gt; <li>一旦合适的细胞被发现，使用ICS微量的负荷三分之一，并将其​​放置在探头。 </li><li>在运行的live CD或预配置的Linux操作系统的个人电脑上，启动命令shell（ 如 bash）的，并在其提示符下键入命令LCG零。这保证了数据采集板被不驱动放大器。 </li><li>申请30 - 50毫巴的正压通过按下一个普通的注射器，通过管道到吸液管保持和，与显微镜的帮助下连接的活塞，将吸管约100微米的片以上。 注:放置吸管的位置，使可直达靶细胞，最好采用显微操作的方法模式。 </li><li>作用在控制电放大器，调整吸移管偏移和输出的测试脉冲（10毫伏）在电压钳模式。 </li><li>通过撤回30毫巴（取决于移液器大小） - 降低压力至10注射器的活塞;轻轻接近细胞并通过观察图像的摄像机监视器上检查一个凹坑的形成。监视测试脉冲，以便随时阻力的增加，通过观看在连接到电放大器，示波器显示的电流波形（或者可以使用命令LCG-密封试验以监测移液器电阻）。 </li><li>释放压力并在必要时施加轻柔负压吸移管，以帮助形成密封时发现的增加吸液管电阻和细胞上的"凹坑"的形成。 </li><li>而密封形式，逐渐减少控股潜力-70毫伏。 </li><li>一旦千兆欧密封已经获得，确保保持电流为0 - 30 PA。施加负压（吸力）来打破隔膜和建立全细胞构型短脉冲。或者，你可以注入电压和强短脉冲（ <e米&gt;即使用放大器上的&#39;ZAP&#39;命令或保持细胞在非常负）破裂的膜，这取决于所用的制备和玻璃吸管。 </li><li>切换到电流钳模式，并验证静息电位是典型的健康细胞。对于使用钾葡糖基溶液皮层锥体神经元，该值通常在-65和-75毫伏。 </li></ol>一个神经元的电响应性能4.半自动表征<ol><li>创建一个目录来存储用户的数据。为了做到这一点的脚本包含在LCG的live CD创建基于日期的文件夹中聘用。在命令提示符下使用它，类型 CD〜/实验 LCG创建-实验文件夹-s PSP，in_vivo_like 这将创造一个为单元格中的数据将被保存的文件夹（和&#39;PSP&#39;和&#39;in_vivo_like"子文件夹），它会打印它的名字到终端窗口;它也可以存储其他信息，例如吸液管电阻和使用该脚本的细胞类型。 </li><li>使用命令将目录更改为新创建的文件夹 CD〜/ &lt;文件夹&gt; 文件夹名称是由命令LCG创建-实验文件夹，将有当天的时间戳（ 即年-月-日）显示的一个，因为在20140331A01。 </li><li>确保该放大器被设置在电流钳模式中操作，该电缆连接和放大器的外部电压指令，如果存在的话，被启用。 </li><li>输入命令LCG-ecodeat命令提示符。这需要一系列命令（即LCG-AP，LCG-VI，LCG斜坡，LCG-tau蛋白和LCG步），用于表征细胞的基本反应特性。 LCG-ECODE要求用户指定两个参数:当前的1毫秒长的脉冲用于诱发单个尖峰在细胞的幅度，和当前柱塞的最大振幅p注入盒，发现其rheobase。 使用以下命令语法: LCG-ECODE --pulse振幅X --ramp幅度ÿ 同的值X和Y（以pA）在足以使细胞火响应于1毫秒长的脉冲并持续注入电流的，分别的一个的选择。 注意:这些协议需要以使用活动电极补偿（AEC）15执行"电极内核"的数值的估计。嘈杂的电流注入用于估计内核和系统会提示用户确认样本构成的内核数量。对电极内核的意义以及如何选择内核的样本数量的详细信息，请参阅15。 </li></ol>通过模拟突触和模拟体内般的背景活动5.注射电导<ol><li>注射模拟兴奋性突触后电位<ol><li>更改的目录，你会救下的实验，通过在shell命令提示符下键入以下命令: CD PSP / 01 </li><li>一个LCG配置文件复制到当前目录，并用文本编辑器（纳米在这个例子中）打开它通过键入以下命令在shell命令提示符（此示例配置文件包含在源代码和活CD） : CP〜/ local / src目录/ LCG /配置/ epsp.xml 纳米epsp.xml 注意:这是简单地与连接到彼此不同的实体的文本文件。欲了解更多详情，请参阅代表结果部分。 </li><li>如有必要，编辑inputChannel，outputChannel的inputConversionFactor并在此文件中的outputConversionFactor来匹配用户的设置。 </li><li>通过发出COM计算进行有效电极补偿"使用LCG的方法来执行单电极钳动态"所需的电极内核普通话 LCG内核 这将提示在内核的点数。再次，选择使得电极内核覆盖的指数衰减尾部的端部的数量。 </li><li>使用命令执行动态钳实验 LCG-实验-c epsp.xml </li><li>列出的文件和通过使用命令可视化的结果 ls -l命令 LCG积文件-f最后</li></ol></li><li>注射模拟抑制性突触后电位<ol><li>创建一个文件夹，并通过在shell命令提示符下键入以下命令epsp.xml文件复制到它: MKDIR ../02 CP epsp.xml ../02/ipsp.xml CD ../02 </li><li>通过使用文本编辑器编辑配置文件:改变突触逆转潜力和上升和下降时间的模型突触Exp2Synapse常数以下几点: 参数&gt; &lt;E&gt; -80 &lt;/ E&gt; &lt;tauRise&gt; 0.8E-3 &lt;/ tauRise&gt; &lt;TauDecay&gt; 10E-3 &lt;/ tauDecay&gt; &lt;参数&gt; 退出文本编辑器。 </li><li>计算电极内核和5.1进行实验，通过在shell命令提示符下键入以下命令: LCG内核 LCG-实验-c ipsp.xml </li><li>列表中的文件和可视化的结果，通过在shell命令提示符下键入以下命令: ls -l命令 LCG-情节文件-f &lt;filename.h5&gt; </li></ol></li><li>模拟体内样的背景活动: <ol><li>更改为要保存下面的实验，如前所示，通过在shell命令提示符下键入以下命令目录: CD ../../in_vivo_like/01 </li><li>复制配置文件从LCG源目录，在shell的命令提示符下键入以下命令: CP〜/ local / src目录/ LCG /配置/ in_vivo_like.xml 纳米in_vivo_like.xml <li>调整DAQ配置参数，为用户的设置，如5.1.3节并退出编辑器。 </li><li>计算电极内核和5.1进行实验，通过在shell命令提示符下键入以下命令: LCG内核 LCG-实验-c in_vivo_like.xml -n 10 -i 3 该&#39;-n 10&#39;和&#39;-i 3&#39;开关指示的刺激应重复10次在3秒的时间间隔。 </li><li>可视化的原始痕迹通过在shell命令提示符下使用以下命令: LCG积文件-f所有</li></ol></li></ol>

<ol>
	<li>Saleem, A. B., Ayaz, A., Jeffery, K. J., Harris, K. D., Carandini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+visual+motion+and+locomotion+in+mouse+visual+cortex.">Integration of visual motion and locomotion in mouse visual cortex.</a> Nature neuroscience. 16, 1864-1869 (2013).</li><li>Ahrens, M. B., Li, J. 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=Brain-wide+neuronal+dynamics+during+motor+adaptation+in+zebrafish.">Brain-wide neuronal dynamics during motor adaptation in zebrafish.</a> Nature. 485 (7399), 471-477 (2012).</li><li>Paz, J. T., Davidson, T. 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=Closed-loop+optogenetic+control+of+thalamus+as+a+tool+for+interrupting+seizures+after+cortical+injury.">Closed-loop optogenetic control of thalamus as a tool for interrupting seizures after cortical injury.</a> Nature neuroscience. 16 (1), 64-70 (2013).</li><li>Wallach, A., Eytan, D., Gal, A., Zrenner, C., Marom, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+response+clamp.">Neuronal response clamp.</a> Frontiers in neuroengineering. 3 (April), 3 (2011).</li><li>Linaro, D., Couto, J., Giugliano, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Command-line+cellular+electrophysiology+for+conventional+and+real-time+closed-loop+experiments.">Command-line cellular electrophysiology for conventional and real-time closed-loop experiments.</a> Journal of neuroscience. 230, 5-19 (2014).</li><li>Sharp, A., O&#8217;Neil, M., Abbott, L. F., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+clamp:+computer-generated+conductances+in+real+neurons.">Dynamic clamp: computer-generated conductances in real neurons.</a> Journal of neurophysiology. 69 (3), 992-995 (1993).</li><li>Robinson, H. P., Kawai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+of+digitally+synthesized+synaptic+conductance+transients+to+measure+the+integrative+properties+of+neurons.">Injection of digitally synthesized synaptic conductance transients to measure the integrative properties of neurons.</a> Journal of neuroscience methods. 49 (3), 157-165 (1993).</li><li>Vervaeke, K., Hu, H., Graham, L. J., Storm, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrasting+effects+of+the+persistent+Na++current+on+neuronal+excitability+and+spike+timing.">Contrasting effects of the persistent Na+ current on neuronal excitability and spike timing.</a> Neuron. 49 (2), 257-270 (2006).</li><li>White, J. A., Klink, R., Alonso, A., Kay, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise+from+voltage-gated+ion+channels+may+influence+neuronal+dynamics+in+the+entorhinal+cortex.">Noise from voltage-gated ion channels may influence neuronal dynamics in the entorhinal cortex.</a> Journal of neurophysiology. 80 (1), 262-269 (1998).</li><li>Destexhe, a., Rudolph, M., Fellous, J. M., Sejnowski, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuating+synaptic+conductances+recreate+in+vivo-like+activity+in+neocortical+neurons.">Fluctuating synaptic conductances recreate in vivo-like activity in neocortical neurons.</a> Neuroscience. 107 (1), 13-24 (2001).</li><li>Fellous, J. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Persistent+Activity+by+Background+Inhibition+in+an+In+Vitro+Model+of+a+Cortical+Microcircuit.">Regulation of Persistent Activity by Background Inhibition in an In Vitro Model of a Cortical Microcircuit.</a> Cerebral Cortex. 13 (11), 1232-1241 (2003).</li><li>Gal, A., Eytan, D., Wallach, A., Sandler, M., Schiller, J., Marom, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+excitability+over+extended+timescales+in+cultured+cortical+neurons.+The+Journal+of+neuroscience.">Dynamics of excitability over extended timescales in cultured cortical neurons. The Journal of neuroscience.</a> the official journal of the Society for Neuroscience. 30 (48), 16332-16342 (2010).</li><li>Wang, Y., Toledo-Rodriguez, 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=Anatomical,+physiological+and+molecular+properties+of+Martinotti+cells+in+the+somatosensory+cortex+of+the+juvenile+rat.">Anatomical, physiological and molecular properties of Martinotti cells in the somatosensory cortex of the juvenile rat.</a> The Journal of physiology. 561 (Pt 1), 65-90 (2004).</li><li>Wang, Y., Gupta, A., Toledo-Rodriguez, M., Wu, C. Z., Markram, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical,+physiological,+molecular+and+circuit+properties+of+nest+basket+cells+in+the+developing+somatosensory+cortex).">Anatomical, physiological, molecular and circuit properties of nest basket cells in the developing somatosensory cortex).</a> Cerebral cortex (New York, N.Y). 12 (4), 395-410 (1991).</li><li>Brette, R., Piwkowska, Z., 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=High-resolution+intracellular+recordings+using+a+real-time+computational+model+of+the+electrode.">High-resolution intracellular recordings using a real-time computational model of the electrode.</a> Neuron. 59 (3), 379-391 (2008).</li><li>Rutishauser, U., Kotowicz, A., Laurent, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+closed-loop+presentation+of+sensory+stimuli+conditional+on+the+internal+brain-state+of+awake+animals.">A method for closed-loop presentation of sensory stimuli conditional on the internal brain-state of awake animals.</a> Journal of neuroscience. 215 (1), 139-155 (2013).</li><li>Margrie, T., Brecht, M., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo,+low-resistance,+whole-cell+recordings+from+neurons+in+the+anaesthetized+and+awake+mammalian+brain.">In vivo, low-resistance, whole-cell recordings from neurons in the anaesthetized and awake mammalian brain.</a> Pflugers Archiv European Journal of Physiology. 444 (4), 491-498 (2002).</li><li>Graham, L., Schramm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Dynamic-Clamp+Manipulation+of+Extrinsic+and+Intrinsic+Conductances:+Functional+Roles+of+Shunting+Inhibition+and+I+BK+in+Rat+and+Cat+Cortex.">In Vivo Dynamic-Clamp Manipulation of Extrinsic and Intrinsic Conductances: Functional Roles of Shunting Inhibition and I BK in Rat and Cat Cortex.</a> Dynamic Clamp: From Principles to Applications. , (2008).</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=.">.</a> Single-channel recording. , (1995).</li><li>Molleman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Patch Clamping. , (2002).</li><li>Davie, J. T., Kole, M. H. P., 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=Dendritic+patch-clamp+recording.">Dendritic patch-clamp recording.</a> Nature Protocols. 1 (3), 1235-1247 (2006).</li><li>Gold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Axon Guide for Electrophysiolog., &amp; Biophysics Laboratory Techniques... , (2007).</li><li>Mainen, Z. F., Sejnowski, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+of+spike+timing+in+neocortical+neurons.">Reliability of spike timing in neocortical neurons.</a> Science. 268 (5216), 1503-1506 (1995).</li><li>Buzs&#225;ki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+potential+threshold+of+hippocampal+pyramidal+cells+in+vivo+is+increased+by+recent+spiking+activity.">Action potential threshold of hippocampal pyramidal cells in vivo is increased by recent spiking activity.</a> Neuroscience. 105 (1), 121-130 (2001).</li><li>Koch, C., Segev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Neuronal Modeling: From Synapses to Networks. , (1988).</li><li>Silberberg, G., Markram, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disynaptic+inhibition+between+neocortical+pyramidal+cells+mediated+by+Martinotti+cells.">Disynaptic inhibition between neocortical pyramidal cells mediated by Martinotti cells.</a> Neuron. 53 (5), 735-746 (2007).</li><li>Berger, T. K., Silberberg, G., Perin, R., Markram, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+bursts+self-inhibit+and+correlate+the+pyramidal+network.">Brief bursts self-inhibit and correlate the pyramidal network.</a> PLoS biology. 8 (9), (2010).</li><li>Tsodyks, M., Pawelzik, K., Markram, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+networks+with+dynamic+synapses.">Neural networks with dynamic synapses.</a> Neural computation. 10 (4), 821-835 (1998).</li><li>Kapfer, C., Glickfeld, L. L., Atallah, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supralinear+increase+of+recurrent+inhibition+during+sparse+activity+in+the+somatosensory+cortex.">Supralinear increase of recurrent inhibition during sparse activity in the somatosensory cortex.</a> Nature. 10 (6), 743-753 (2007).</li></ol>

The authors have nothing to disclose.

在该文本的完整协议的实时实施，闭环单细胞电生理实验进行了说明，使用膜片钳技术和最近开发的软件工具箱称为LCG。要优化录音的质量是至关重要的录音设置正确接地，屏蔽和无振动:这可确保稳定持久的全细胞进入细胞，这与自动化的刺激方案整个路段的可能性在一起允许对实验的吞吐量最大化。 两种情况，即LCG可以应用已经提出，一个单元的即在其电生理特性（ <stron...

<html>近年来，细胞电已从在电压和电流钳实验采用现代闭环协议传统的开环模式演变而来。最有名的闭环技术也许是动态钳6,7，这使合成注射人工电压门控离子通道，以确定神经元膜电压8中的非确定性闪烁的影响的深入研究离子通道在神经反应动力学9，以及娱乐的现实体外接近体内的突触背景活动10。 已经提出的其它闭环范例包括反应性夹具11，以研究在体外自我维持的持久性活动的生成，并且响应夹紧4,12，调查细胞机制底层神经元兴奋。 内容】"&gt;这里，我们描述了允许应用各种闭环电协议中的急性脑切片进行全细胞膜片钳记录的上下文中，功能强大的框架。我们将展示如何通过膜片钳记录装置来记录体膜电压从幼年大鼠的躯体感觉皮层和锥体神经元使用申请LCG，在理论和神经生物学的神经工程实验室开发的一个命令行的软件工具箱三种不同的闭环协议。 简要地，所描述的协议中，首先在自动注射的一系列电流钳刺激波，相关的一大组有源和无源膜性能的表征。这些已被建议来捕获的细胞的电生理学表型在其响应性质方面至刻板一系列刺激波形。称为小区的电子代码 （ 例如 ，参见＆＃160; 13,14），电反应这样的集合所使用的几个实验室的它们的电学性能的基础上，以客观地分类的神经元。这包括在固定输入输出传递关系（FI曲线），通过创新的技术，包括通过一个比例 - 积分 - 微分（PID）控制器的装置触发速率的闭环，实时控制的分析的，现实的体内样背景突触活动的第二体外制剂10和，由虚拟GABA能interneuron，这是由计算机模拟的装置在两个同时记录锥体细胞的实时第三人工连接的娱乐。 此外，LCG实现了被称为活跃的补偿电极（AEC）15，它允许使用实现单电极钳动态协议技术。这使得补偿的不良影响（一当它被用于输送细胞内的刺激出现的记录电极的rtifacts）。该方法是基于在记录电路的等效电特性的非参数估计。 在本文中描述的技术和实验方案可以在常规开环电压和电流钳实验可容易地提供，并且可以延伸到其他制剂，如胞外4,16或细胞内记录体内 17,18。设置为全细胞膜片钳电的精心组装是稳定的，高品质的录音非常重要的一步。在下文中，我们假设这样一个实验装置已经提供给实验者，和我们的注意力集中于描述LCG的使用。请读者指出，19-22关于优化和调试的其他提示。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Tissue slicer</td>
			<td>Leica</td>
			<td>VT-1000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette puller</td>
			<td>Sutter</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>WPI</td>
			<td>1B150F-4</td>
			<td>1.5/0.84 mm OD/ID, with filament</td>
		</tr>
		<tr>
			<td>Vibration isolation table</td>
			<td>TMC</td>
			<td>20 Series</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>DMLFS</td>
			<td>40X Immersion Objective</td>
		</tr>
		<tr>
			<td>Manipulators</td>
			<td>Scientifica</td>
			<td>PatchStar</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifiers</td>
			<td>Axon Instruments</td>
			<td>MultiClamp 700B</td>
			<td>Computer controlled</td>
		</tr>
		<tr>
			<td>Data acquisition card</td>
			<td>National Instruments</td>
			<td>PCI-6229</td>
			<td>Supported by Comedi Linux Drivers</td>
		</tr>
		<tr>
			<td>Desktop computer</td>
			<td>Dell</td>
			<td>Optiplex 7010 Tower</td>
			<td>OS: real-time Linux</td>
		</tr>
		<tr>
			<td>Oscilloscopes</td>
			<td>Tektronix</td>
			<td>TDS-1002</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion Pump</td>
			<td>Gibson</td>
			<td>MINIPULS3</td>
			<td>Used with R4 Pump head (F117606)</td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>Multichannel Systems</td>
			<td>TC02</td>
			<td>PH01 Perfusion Cannula</td>
		</tr>
		<tr>
			<td>Manometer</td>
			<td>Testo</td>
			<td>510</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Memmert</td>
			<td>WB14</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>71376</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td>ACSF, ICS</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma</td>
			<td>S3139</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S6014</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7528</td>
			<td>ACSF</td>
		</tr>
		<tr>
			<td>K-Gluconate</td>
			<td>Sigma</td>
			<td>G4500</td>
			<td>ICS</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td>ICS</td>
		</tr>
		<tr>
			<td>Mg-ATP</td>
			<td>Sigma</td>
			<td>A9187</td>
			<td>ICS</td>
		</tr>
		<tr>
			<td>Na2-GTP</td>
			<td>Sigma</td>
			<td>51120</td>
			<td>ICS</td>
		</tr>
		<tr>
			<td>Na2-Phosphocreatine</td>
			<td>Sigma</td>
			<td>P7936</td>
			<td>ICS</td>
		</tr>
	</tbody>
</table>

real-time electrophysiology: using closed-loop protocols to probe neuronal dynamics and beyond

实验神经学正在经历的发展和应用新颖的和往往是复杂的，闭环协议，其中的刺激施加实时取决于系统的响应的兴趣增加。最近的应用范围从虚拟现实系统的实施为研究运动 ​​反应无论是在小鼠1和2斑马鱼，以控制使用以下3光遗传学皮质中风发作。闭环技术的关键优点在于，探测更高维属性不能直接访问或依赖于多个变量，如神经元兴奋4和可靠性，而在同一时间最大化实验吞吐量的能力。在这方面的贡献，并在细胞电的情况下，我们将描述如何各种闭环协议适用于锥体皮层神经元，REC响应特性的研究在从幼年大鼠躯体感觉皮层急性脑片膜片钳技术orded细胞内。由于没有市售或开源软件提供了所有必需的用于高效执行这里所描述的实验的特征，开发了一种新的软件工具箱称为LCG 5，其模块化结构最大化的计算机代码的重用，并且容易实现新的实验范例的实施。刺激波形使用的是紧凑元说明中指定和完整实验方案在基于文本的配置文件中描述。此外，LCG具有适合于反复试验和实验规程自动化的命令行界面。

在前面的章节中，我们已经描述了如何使用该软件工具箱LCG表征L5锥体细胞的电生理特性和重新体内样突触活性的切片准备。使用命令行界面和半自动化协议的有利于实验，这可能对所产生的数据的输出和质量有很大的影响的再现性和效率。另外，由于数据被保存在一个一致的方式，很容易扩展分析，以一个特定的目标。 图1示出了一个实验，其中使用六个不同的协议的小区的?...

Watch this Scientific Journal Video about Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond at JoVE.com

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

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

实时电:采用闭环协议来探测神经动力学和超越

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

real-time-electrophysiology-using-closed-loop-protocols-to-probe

Research

JoVE Journal

Neuroscience

11.4K 次观看.  University of Antwerp. Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board. 

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

An Experimental Platform to Study the Closed-loop Performance of Brain-machine Interfaces

The non-stationary nature and variability of neuronal signals is a fundamental problem in brain-machine interfacing. We developed a brain-machine interface to assess the robustness of different control-laws applied to a closed-loop image stabilization task. Taking advantage of the well-characterized fly visuomotor pathway we record the electrical activity from an identified, motion-sensitive neuron, H1, to control the yaw rotation of a two-wheeled robot. The robot is equipped with 2 high-speed video cameras providing visual motion input to a fly placed in front of 2 CRT computer monitors. The activity of the H1 neuron indicates the direction and relative speed of the robot's rotation. The neural activity is filtered and fed back into the steering system of the robot by means of proportional and proportional/adaptive control. Our goal is to test and optimize the performance of various control laws under closed-loop conditions for a broader application also in other brain machine interfaces.

We use a closed-loop fly-machine interface to investigate general principles in neuronal control.

研究脑机接口的闭环性能的实验平台

The non-stationary nature and variability of neuronal signals is a fundamental problem in brain-machine interfacing. We developed a brain-machine ...

科研

神经科学

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

器官型培养的神经元雪崩多电极阵列记录

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

同时脑电图，实时测量的啮齿动物的大脑皮质中的神经元活动的乳酸浓度与Optogenetic的操作

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

作为一种工具来描绘分子途径参与神经毒性和神经保护神经元细胞在线实时阻抗为基础的细胞分析仪

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

Closed-loop Neuro-robotic Experiments to Test Computational Properties of Neuronal Networks

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation of a given stimulus might be dependent on context and history. If this is actually the case, bi-directional interactions between the brain (or if need be a reduced model of it) and sensory-motor system can shed a light on how encoding and decoding of information is performed. Here an experimental system is introduced and described in which the activity of a neuronal element (i.e., a network of neurons extracted from embryonic mammalian hippocampi) is given context and used to control the movement of an artificial agent, while environmental information is fed back to the culture as a sequence of electrical stimuli. This architecture allows a quick selection of diverse encoding, decoding, and learning algorithms to test different hypotheses on the computational properties of neuronal networks.

In this paper, an experimental framework to perform closed-loop experiments is presented, in which information processing (i.e., coding and decoding) and learning of neuronal assemblies are studied during the continuous interaction with a robotic body.

闭环神经机器人实验来检验神经网络的计算性能

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

对于电生理和钙成像急性神经组织的长时间培养

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

测定在运动神经元样NSC-34细胞中实时测量核细胞质运输

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 &#181;m away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

用四甲基铵进行实时离子电渗疗法，以量化体细胞分数和脑细胞外空间的变性

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

实时 cAMP 检测气味受体激活的活细胞测量

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

Using Neuron Spiking Activity to Trigger Closed-Loop Stimuli in Neurophysiological Experiments

Closed-loop neurophysiological systems use patterns of neuronal activity to trigger stimuli, which in turn affect brain activity. Such closed-loop systems are already found in clinical applications, and are important tools for basic brain research. A particularly interesting recent development is the integration of closed-loop approaches with optogenetics, such that specific patterns of neuronal activity can trigger optical stimulation of selected neuronal groups. However, setting up an electrophysiological system for closed-loop experiments can be difficult. Here, a ready-to-apply Matlab code is provided for triggering stimuli based on the activity of single or multiple neurons. This sample code can be easily modified based on individual needs. For instance, it shows how to trigger sound stimuli and how to change it to trigger an external device connected to a PC serial port. The presented protocol is designed to work with a popular neuronal recording system for animal studies (Neuralynx). The implementation of closed-loop stimulation is demonstrated in an awake rat.

This protocol demonstrates how to use an electrophysiological system for closed-loop stimulation triggered by neuronal activity patterns. Sample Matlab code that can be easily modified for different stimulation devices is also provided.

在神经生理学实验中利用神经元刺激活性触发闭环刺激

Closed-loop neurophysiological systems use patterns of neuronal activity to trigger stimuli, which in turn affect brain activity. Such closed-loop systems ...