作者感谢奥弗·费纳曼，弗雷德·沃尔夫，梅纳姆·塞加尔，安德烈亚斯·尼夫和艾塔恩·雷威尼非常有益的讨论。作者感谢伊兰·布雷斯金和佐迪·索利安诺开发技术的早期版本。作者感谢茨维·拉斯蒂和吉恩·皮尔·埃克曼与理论概念的帮助。这项研究是由密涅瓦基金会，科技，以色列，部和以色列科学基金会授予1320年至1309年和碧国家科学基金会资助2008331支持。

伦理声明:涉及动物的处理程序是按照魏茨曼科学研究所的机构动物护理和使用委员会（IACUC），以及适当的以色列法律的指引完成。魏兹曼研究所由协会为实验动物国际评估和认可委员会（AAALAC）的认可。魏茨曼机构动物护理和使用委员会批准了这项研究，与海马神经元进行。 1.二维（2D）和一维（1D）海马培养制备<ol><li>盖玻片2D培养的制备。 <ol><li>制备平板培养基（PM）组成:0.9毫升极限必需培养基（MEM）+ 3G，0.05毫升胎牛血清（FCS），0.05毫升热灭活的马血清（HI HS）和B27添加剂的1μL。注意:MEM + 3G包含每500毫升MEM的X 1，1 mL庆大霉素，5毫升稳定的L-谷氨酰胺的100倍（见<stro纳克&gt;材料/试剂）和5毫升60％D的表- （+） -葡萄糖。 </li><li>制备组成硼酸盐缓冲液:在200mL双蒸水（DDW）1.9克硼砂（四硼酸钠十水合物）（在60℃下混合），并在200毫升DDW1.24克硼酸。滴定最终溶液使用的1M HCl pH 8.5中。注意:将最终的溶液为400毫升的0.1M硼酸盐缓冲液。 </li><li>浸泡在65％硝酸的玻璃盖玻片上2小时。冲洗在DDW三次，接着冲洗三次以绝对分析试剂（ABS AR）乙醇。 </li><li>简要地两次或三次通过每个盖玻片通过本生灯的火焰1 - 2秒，然后离开用稀释11 mL聚-L-赖氨酸0.01％的溶液温育在过夜24个孔板:在硼酸盐缓冲液5（ 0.1M，pH值8.4）。然后冲洗盖玻片在DDW三次，并用每孔1mL的PM过夜离开在一个标准的37℃，5％CO 2培养箱中。 </li></ol></li><li>盖玻片1D文化的准备。 <ol><li>清洁摹小姑娘盖玻片浸渍在由75毫升DDW，25毫升25％氨水溶液和25ml 30％过氧化氢的碱溶液食人鱼。与30分钟，然后用氮气干燥的玻璃盖玻片上的加热板的玻璃盖玻片上，在〜50℃的地方溶液。 </li><li>外套盖玻片第一与6层的厚度的薄的铬膜（99.999％），其次是金（99.999％）30的层，即使用蒸汽或溅射沉积。 <ol><li>为了实现0.05的溅射速率 - 1埃/秒使用的溅射机2电子束，并用目标尺寸2&quot; 260升/秒的真空系统和4&quot; 。使用可以从0旋转舞台 - 100转每分钟（RPM）。使用的直流（DC）溅射的0功率 - 750瓦。 </li><li>使用30rpm的旋转和氩99.999％在腔室10毫托的压力来获得血浆。 </li><li>操作所述溅射枪的电源在40瓦的DC铬溅射功率，导致〜0.12埃/秒的覆盖率，并且在10瓦的直流溅射功率为金，导致〜0.28埃/秒。 </li></ol></li><li>在使用超声30分钟100mL的ABS AR乙醇将0.1g 1-十八烷硫醇。放置2个小时铬 - 金涂覆的盖玻片在该溶液中，然后用乙醇ABS AR，并用氮气干燥洗​​净。 </li><li> 700rpm下-在600 2小时-制备100毫升的Dulbecco氏磷酸盐缓冲盐水（D-PBS）和3.5克三嵌段共聚合物的溶液搅拌1（参见材料/试剂的表 ）。放置盖玻片在1个小时的溶液中。干盖玻片用氮气。 </li><li>通过刮擦生物排斥层22机械地蚀刻所期望的图案。做到这一点使用笔式绘图仪，其中，所述笔通过蚀刻针代替。划伤通过金属层的图案，以达到下面的玻璃。由计算机控制该过程以实现复制所需的图案。通过该方法形成的图案展示<html> D IN 图2和图3。 </li><li>制备100毫升的D-PBS，3.5克三嵌段共聚物，35.7μL/毫升纤连蛋白和29微升/毫升的层粘连蛋白生物相容层。 <ol><li>消毒在紫外光盖玻片至少10分钟。孵育在制备生物兼容的溶液中过夜盖玻片。 注:生物相容层将形成仅发生在生物排斥层已在先前步骤中被蚀刻掉。 </li><li>第二天洗具P-DBS盖玻片两次。在PM孵育盖玻片过夜。盖玻片现在已经准备好电池电镀。 </li></ol></li></ol></li><li>根据已广泛前面23，24日公布的标准程序进行清扫。 <ol><li>简言之，提取海马或皮质从大鼠胚胎，通常在一天E19，或从小鼠中，通常在一天E17 23，类= "外部参照"&gt; 24。 </li><li>离解的细胞首先在20木瓜蛋白酶溶液- 30分钟，随后通过机械研磨24与玻璃吸管的尖部是火抛光。 注意:如果落入细胞从遗传修饰的小鼠然后从每个胚组织应在一个单独的1.5毫升的塑料管在整个过程中得以保持。 </li><li>播种前用计数台盼蓝细胞。 注:对于转基因动物的计数应分别对每个胚胎完成。 </li><li>对于以每孔85万个细胞2D培养，种子小鼠的神经元在750000个大鼠神经元。对于图1D，种子以每孔65万个细胞。播种，确保盖玻片均匀覆盖后小幅立即摇板。 </li></ol></li><li>在神经细胞的维护。 <ol><li>制备变换媒体（每毫升）组成的（CM）:0.9毫升MEM + 3G，0.1毫升HI HS，10μL5-氟-2&#39;-脱氧尿苷（FUDR）与尿苷100X &lt;/ LI&gt; <li>制备（每毫升）组成的最终培养基（FM）:0.9毫升MEM + 3G和0.1毫升HI HS。 </li><li>后在体外 4天（DIV）替换1-1.5毫升CM PM。在6 DIV，更换新鲜的CM CM的50％。在DIV 8，改变介质至1.5mL FM，随后调频的每2天有50％的变化。大约一个星期后自发同步活动出现。 </li></ol></li><li>与荧光染料神经细胞自发或诱发活动的影像。 <ol><li>制备50微克钙敏感的荧光染料的溶液（参见材料/试剂的表 ）的50微升DMSO（二甲亚砜）。 </li><li>制备含有（以mM计）10个HEPES，4氯化钾，细胞外记录溶液（EM）2 CaCl 2的，1 MgCl 2的，139的NaCl，10 D-葡萄糖，蔗糖45（pH 7.4）中。 </li><li>孵育神经元培养物在2mL EM用1个小时的钙敏感的荧光染料溶液的8μL。避光保存，并轻轻转动，以确保homogen染料对细胞的OU中蔓延。 </li><li>更换成像前用新鲜EM溶液。荧光成像是体现在图4中 。 </li><li>图像中的与钙荧光成像（在488nm的激发峰，在520nm的发射峰）的滤光器的荧光显微镜，使用照相机和软件能够视场内定量的关注区域（ROI）的任何区域的强度的显微镜。 </li></ol></li></ol>文化2.电刺激注:电刺激基本设置示于图1。在其上神经元培养物已生长约14天盖玻片放置在陪替氏培养皿在荧光显微镜下。神经元的电活动使用钙敏感性染料进行成像而将电压经由两对浴电极，其定位在培养外部施加。电极由作为驱动其输出由双通道放大器放大的点火发生器。因为电场矢量是直接确定的，所以用于刺激的电压控制优于更标准的电流控制器25,26 ，因此能够直接的矢量相加和组合。这需要仔细检查电场的均匀性，这可以在电压控制的情况下在整个样品上进行。当使用电压控制时，应注意避免任何接地回路，并确认电场的均匀性（见下文2.2）。 <ol><li>对于均匀电场的电刺激，使用一对平行电极线。 <ol><li>使用厚度在0.005"（127μm）左右的由铂制成的电极。当与13mm盖玻片一起使用时，请确保两个电极之间的距离约为11 mm，并将电极定位的1毫米培养上方。 注意:为了使电极保持器（ 图5A和6A）使用聚四氟乙烯（PTFE）。通过PTFE钻窄孔中插入电极。该装置应比细胞外液中较高，使得顶端，其中电极被暴露，将永远不会在与溶液接触来。绝缘，使用环氧树脂胶上可能被露出的电极引线的任何部分。 </li><li>使用方形脉冲形状具有50％的占空比，没有DC分量，以避免电解。改变脉冲10微秒和4毫秒之间的持续时间，以使有效的刺激不燃烧的培养。确保振幅在±22 V（ 见图5）的顺序。方形脉冲可以并联连接到电极的示波器来观察。 注:对于任何所需的波形容易编程，使用商业波形编辑软件（见材料清单 </str翁&gt;）。图形输入所需的波形，并将其发送到波形发生器。 </li></ol></li><li>为了测试场均匀性使用的探针电极。使用的至少1mm×1mm的栅格，以允许探针在电极之间的区域移动，并测量电势。 <ol><li>测量电势。使用<img alt="式（1）" src="/files/ftp_upload/54357/54357equation1.jpg" />计算的电场。使用电极作为参比电极中的一个。测量的电场与100微秒之间变化的脉冲持续时间以4毫秒（ 见图5B为100微秒的脉冲持续时间的一个例子）以验证该字段是刺激持续时间的范围内是均匀的。 注意:请参阅图5D为一个测量的电场的均匀性的一个例子，当脉冲持续时间为1毫秒。 </li></ol></li><li>使用2个垂直双浴电极以产生更复杂的电场的形状，并能够定向在不同方向上字段（ 见图6B）。用2对电极的装置将被使用，并在每对电极的信号将在两个单独的示波器来观察。 <ol><li>电极位置1毫米以上培养并以10的距离 - 彼此11毫米。确保两个电极对浮动的（有没有接地连接），并且不通过测量任何组的电极之间的电阻和验证不存在任何的电极之间的短路的具有任何共同点。验证中使用的所有设备，其连接到电极（如示波器，放大器，信号发生器等 ）相对于浮动到地通过检查所有设备是浮动的，并且没有参考电极的正触摸任何接地设备。 </li><li>要改变场的取向，改变供给到使用解析度的两个电极对的电压的振幅PECT彼此（参见图7A）。例如，对于上电极对0°±使用22V的，它们垂直于所述图案和0伏为另一个电极。为45°，使用±在两个对电极15.6 V的没有相位滞后，为15.6 2 15.6 2 = 22 2的振幅矢量和。 </li><li>施加旋转磁场使用正弦电压脉冲的一个单个波形和余弦电压脉冲与所述两对电极一个单个波形，以产生一个固定的振幅旋转电场（参见图6B）。 注意:如可以在图6B中 ，使用正弦波的一个周期与一个电极±22 V和与±22伏在另一个电极的余弦波的一个周期时，可以观察时，矢量和是与旋转电场循环持续时间相同正弦波和余弦波，并与±22伏的振幅</li></ol></li><li>测量和计算克罗恩氏在相同的培养的神经元培养物与树突轴突的AXIe和Rheobase执行以下步骤。 <ol><li>与图案化的薄（170微米的1D培养物），长（10mm）的线在材料/试剂的表中描述和1.5）使用钙敏感性荧光染料钙成像。钙敏感性染料将被应用到培养，并温育45分钟。染料将通过与新鲜的记录溶液替换洗涤。 </li><li>断开网络，以实现对电刺激的直接反应的人口统计，没有突触传递的效果。要做到这一点，应用的荷包牡丹碱的40μM，其阻断γ-氨基丁酸-A的抑制作用（GABA A）受体，组合10 6-氰基-7-硝基喹喔啉-2,3-二酮的μM（CNQX） ，以阻断α-氨基-3-羟基-5-甲基-4-异恶唑（AMPA）和红藻氨酸受体和的（2R）20μM - 氨基-5- phosphonovaleRIC酸（APV），其阻断N-甲基-D-天冬氨酸（NMDA）受体。所述阻断剂将被添加到记录溶液。 </li><li>作为具有变化的100微秒和4毫秒之间的持续时间在2.1和2.3中描述施加方波脉冲。该信号可以在示波器上观察到。 </li><li>使用荧光显微镜图像采集程序（见1.5）来监视钙瞬变的强度在几个感兴趣区，每个包含几百神经元。获得使用灵敏EMCCD照相机（见材料清单）的图像中，能够至少每秒20帧的图像采集速率。光强度的变化是正比于刺激神经元的数量。估计使用在强度的相对变化刺激神经元的分数。 </li><li>对于每个脉冲持续时间（100微秒到4毫秒），改变施加到起始于其中强度没有变化被看作（几伏特）的电压的电极上的电压的振幅，以THE上的电压其中强度由于所施加的电压的变化已经饱和的（高达±22 V）。 注:强度变化将被分布为累积高斯5相对于所施加的电压为刺激的电压脉冲的每个持续时间。 </li><li>适合的强度与每个时间的施加电压的累积高斯分布并从该配合提取的高斯平均。 注:此平均值是到神经元的反应代表电压。 </li><li>在此过程结束时获得每个刺激持续时间的平均电压施加到神经元答复。使用这些对持续时间和强度绘制的强度-持续时间曲线（ 见图7）。 </li></ol></li></ol> 3.文化的磁刺激注意:用于磁刺激的基本设置在图2中被示出。在右上角显示是，用于在神经元图像钙敏感性染料倒置荧光显微镜。磁线圈（蓝色圆圈）同心地定位上方的神经元环培养，（蓝色轮廓）约5mm。在陪替氏培养皿的圆周上的拾波线圈（红圈）监视由磁脉冲感应的电压。在左上角被示出具有5000千伏的电容器电压负载磁刺激（MS）线圈的所测量的动力学，如从拾波线圈一体化。感应电场（对于14mm的环的半径计算），而磁场在蓝色描绘在绿色被描绘。在底部示出了神经元培养物的图像。在左下方是图案化24毫米盖玻片的亮场图像。在白色区域中的神经元。所拍摄的图形由具有不同半径的同心环的文化。在右下角是变焦到所述环中的一个短段，示出了单独的神经元。对于一个规模，环妇女参与发展th为约200μm。 <ol><li>生长的神经元中的圆形环图案为一维培养刺激（如1.2.5中所述蚀刻）。使用钙成像钙敏感的荧光染料（如在第1.5节所描述的）与图案化上薄（170微米）一维培养，长（10mm）的线。 <ol><li>使用圆形磁线圈和定位培养皿下面和同心大约5mm与线圈。使用大约30毫米（内径，46毫米外径）线圈的一个自定义的线圈与由载有5千伏的最高电压自制或商业MS从动L的电感= 90 毫亨 。 </li><li>使用高电流的高电压开关以磁性刺激神经元培养物排出通过导电线圈的高电压和电流。 5千伏-如使用大电容，100 MF的量级上，以获得1的高电压在21所述的磁刺激（MS）可以构建。或者使用市售可用MS（见 材料/试剂的表 ）。 注意:使用0.254毫米厚6.35毫米宽的聚酯涂覆的铜扁线以制造自制线圈21。打开定制的帧，在与环氧玻璃纤维和浇铸绝缘电线（参见材料/试剂的表 ）。可替代地使用可商购的线圈（参见材料/试剂的表 ）。 </li></ol></li><li>使用旋转磁场刺激二维培养。 </li><li>现在，使用旋转磁场刺激二维培养。火TMS与在盘中没有文化，以不同强度，以示与强度线圈读数的线性相关性。 </li><li>接着，在记录钙瞬变与神经元培养物，开始在增加强度的同时录制两者钙瞬变和线圈烧制TMS。起初，网络突发观察到大的钙瞬变，昭ULD不同步回暖线圈TMS尖峰。继续增加强度，在某些点上，钙瞬变开始变得与TMS尖峰同步。可替换地，或另外地，实现了同步的响应之后，开始降低强度，直到同步取消，以确定所述阈值TMS。 注:条件应保持严格固定的每个设置的，每次使用的确切数量，同样的船只和详细的线圈的位置和方向。 <ol><li>装入记录盘的基座上的拾波线圈，以便它是在平行于神经元培养物的平面和在相对于该培养的固定位置。 注意:这确保了相对于所述磁铁的拾波线圈的定位的依赖性是忠实于培养物的位置，并且在定位的任何差异将在拾波线圈的读数可以忽略不计。 <ol><li>使用两个独立的线圈是位于相互垂直的（ 图8B），以产生旋转磁场和感应电场。每个线圈连接到其自己的MS。确保刺激物以90度（ 图10A）的相位滞后放电类似的电流，产生旋转磁场以〜270 V / m的最大字段（ 图10B）扫描270度的实际空间。 </li><li>的神经元培养物填充有外部记录溶液（EM）的球形玻璃容器内侧的位置。 </li><li>如在步骤中描述2.4.4监测刺激与照相机（在神经元中的荧光强度变化）。 </li><li>在交叉线圈（ 图8B）的情况下，定位拾波线圈并联，并在神经元培养物下面的特定距离。小心地维持整个实验的这种配置。 </li></ol></li></ol></li><li>计算分析一维configura电场用E 最大 = K 1 BR，其中E max是感应电场的最大振幅并且沿着半径为r的环的切线指向灰。 B为磁脉冲的幅度，而k 1是可以使用拾波线圈（ 图8A）进行测定的尺寸比例常数。 </li><li>使用数值模拟软件包（参见物料清单），数值模拟电场21。 </li></ol>

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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=Axon+diodes+for+the+reconstruction+of+oriented+neuronal+networks+in+microfluidic+chambers.">Axon diodes for the reconstruction of oriented neuronal networks in microfluidic chambers.</a> Lab Chip. 11 (21), 3663-3673 (2011).</li><li>Renault, 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=Combining+microfluidics,+optogenetics+and+calcium+imaging+to+study+neuronal+communication+in+vitro.">Combining microfluidics, optogenetics and calcium imaging to study neuronal communication in vitro.</a> PLoS One. 10 (4), e0120680 (2015).</li><li>Roth, B. J., Basser, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+of+the+stimulation+of+a+nerve+fiber+by+electromagnetic+induction.">A model of the stimulation of a nerve fiber by electromagnetic induction.</a> IEEE Trans Biomed Eng. 37 (6), 588-597 (1990).</li></ol>

作者宣称，他们没有竞争的经济利益。

1D图案是可用于多种应用的重要工具。例如，我们已经使用一维图案用于从神经元培养29创建逻辑门以及最近测量大鼠海马神经元5的时值和Rheobase，和唐氏综合征的海马神经元电活动的信号传播速度的下降的减速比野生型（WT）海马神经元27。一维图案的建议的方案是稳健的，这是容易形成任何所需的图案。我们建议观察1D培养前测量，以了解?...

神经元和外部电场之间的相互作用具有根本性的影响以及实际的。虽然人们自从沃尔的倍外部施加的电场可以激发组织已知的，负责神经细胞中生产的所得动作电位的机制最近才开始被揭开1，2，3，4。这包括关于查找导致膜电位的去极化，膜性质和离子通道的作用，甚至该区域响应该电场2,5神经元的机制问题的答案。治疗神经刺激6，7，8，9， <sup10类方法特别依赖于这些信息，这对于针对受影响的地区和了解治疗结果来说可能至关重要。这种理解也可以帮助制定治疗方案和刺激大脑不同区域的新方法。 测量体内大脑内的相互作用为这一理解提供了重要的组成部分，但受到颅骨内测量不精确和低可控性的阻碍。相比之下，文化中的测量可以很容易地以高精度，优异的信噪比和高重现性和控制的高精度执行。使用文化可以阐明集体网络行为的各种神经元属性11,12,13,14 ， </sup&gt; 15，16 。类似地，期望这种良好控制的系统在阐明其他刺激方法的作用机制方面是非常有效的，例如在光激活神经元17,18,19中的光学刺激期间如何通道开放负责产生动作电位。 这里的重点是描述可以通过外部电场有效地激发神经元的工具的开发和理解。在本文中，我们描述了二维和一维图案海马培养物的制备，使用浴电极直接施加的电场的不同构型和取向的刺激，最后通过一个刺激二维和图案化的一维培养物时变磁场，其引起电场5，20，21 。

<table><tbody><tr><td>APV</td><td>Sigma-Aldrich</td><td>A8054</td><td>Disconnect the network. Mentioned in Section 2.4.2</td></tr><tr><td>B27 supp</td><td>Gibco</td><td>17504-044</td><td>Plating medium. Mentioned in Section 1.1.1</td></tr><tr><td>bicuculline</td><td>Sigma-Aldrich</td><td>14343</td><td>Disconnect the network. Mentioned in Section 2.4.2</td></tr><tr><td>Borax (sodium tetraborate decahydrate)</td><td>Sigma-Aldrich</td><td>S9640</td><td>Borate buffer. Mentioned in Section 1.1.2</td></tr><tr><td>Boric acid</td><td>Frutarom LTD</td><td>5550710</td><td>Borate buffer. Mentioned in Section 1.1.2</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; , 1 M</td><td>Fluka&amp;nbsp;</td><td>21098</td><td>Extracellular recording solution. Mentioned in Section 1.5.2</td></tr><tr><td>CNQX</td><td>Sigma-Aldrich</td><td>C239</td><td>Disconnect the network. Mentioned in Section 2.4.2</td></tr><tr><td>COMSOL</td><td>COMSOL Inc</td><td>Multiphysics 3.5</td><td>Numerical simulation. Mentioned in Section 3.5.2</td></tr><tr><td>D-(+)-Glucose, 1 M</td><td>Sigma-Aldrich</td><td>65146</td><td>Plating medium, Extracellular recording solution. Mentioned in Sections 1.1.1 and 1.5.2</td></tr><tr><td>D-PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td>Cell Cultures. Mentioned in Sections 1.2.4 and 1.2.6</td></tr><tr><td>FCS (FBS)</td><td>Gibco</td><td>12657-029</td><td>Plating medium. Mentioned in Section 1.1.1</td></tr><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>F1141</td><td>Bio Coating. Mentioned in Section 1.2.6</td></tr><tr><td>Fluo4AM</td><td>Life technologies</td><td>F14201</td><td>Imaging of spontaneous or evoked activity. Mentioned in Sections 1.5.1, 1.5.3, and 1.5.5</td></tr><tr><td>FUDR</td><td>Sigma-Aldrich</td><td>F0503</td><td>Changing medium. Mentioned in Section 1.4.1</td></tr><tr><td>Gentamycin</td><td>Sigma-Aldrich</td><td>G1272</td><td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1</td></tr><tr><td>GlutaMAX 100x</td><td>Gibco</td><td>35050-038</td><td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1</td></tr><tr><td>Hepes, 1 M</td><td>Sigma-Aldrich</td><td>H0887</td><td>Extracellular recording solution. Mentioned in Section 1.5.2</td></tr><tr><td>HI HS</td><td>BI</td><td>04-124-1A</td><td>Plating medium, Changing medium, Final medium. Mentioned in Sections 1.1.1, 1.4.1, and 1.4.2</td></tr><tr><td>KCl,&amp;nbsp; 3 M</td><td>Merck</td><td>1049361000</td><td>Extracellular recording solution. Mentioned in Section 1.5.2</td></tr><tr><td>Laminin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>L2020</td><td>Bio Coating. Mentioned in Section 1.2.6</td></tr><tr><td>MEM x 1</td><td>Gibco</td><td>21090-022</td><td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.4.1&amp;nbsp;&amp;nbsp;&amp;nbsp; 1.4.2</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; , 1 M</td><td>Sigma-Aldrich</td><td>M1028</td><td>Extracellular recording solution. Mentioned in Section 1.5.2</td></tr><tr><td>NaCl, 4 M</td><td>Bio-Lab</td><td>19030591</td><td>Extracellular recording solution. Mentioned in Section 1.5.2</td></tr><tr><td>Octadecanethiol</td><td>Sigma-Aldrich</td><td>01858</td><td>Cleaning Cr-Au coated coverslips (1D cultures). Mentioned in Section 1.2.3</td></tr><tr><td>Pluracare F108 NF Prill</td><td>BASF Corparation&amp;nbsp;</td><td>50475278</td><td>Bio-Rejection Coating, Bio Coating. Mentioned in Sections 1.2.4 and 1.2.6</td></tr><tr><td>Poly-L-lysine 0.01% solution&amp;nbsp;</td><td>Sigma-Aldrich</td><td>&amp;nbsp;P47075</td><td>Promote cell division. Mentioned in Section 1.1.4</td></tr><tr><td>Sucrose, 1 M</td><td>Sigma-Aldrich</td><td>S1888</td><td>Extracellular recording solution. Mentioned in Section 1.5.2</td></tr><tr><td>Thiol&amp;nbsp;</td><td>Sigma-Aldrich</td><td>1858</td><td>Bio-Rejection Coating. Mentioned in Section 1.2.3</td></tr><tr><td>URIDINE</td><td>Sigma-Aldrich</td><td>U3750</td><td>Changing medium. Mentioned in Section 1.4.1</td></tr><tr><td>Sputtering machine</td><td>AJA International, Inc</td><td>ATC Orion-5Series&amp;nbsp;</td><td>coating glass with thin layers of metal. Mentioned in Section 1.2.2</td></tr><tr><td>Pen plotter&amp;nbsp;</td><td>Hewlett Packard&amp;nbsp;</td><td>HP 7475A</td><td>Etching of pattern to the coated coverslip. Mentioned in Section 1.2.5</td></tr><tr><td>Electrodes wires&amp;nbsp;</td><td>A-M Systems, Carlsborg WA</td><td>767000</td><td>Electric stimulation of neuronal cultures. Mentioned in Sections 2.1, 2.2, 2.3, and 2.4.5</td></tr><tr><td>Signal generator</td><td>BKPrecision</td><td>4079</td><td>Shaping of the electric signal. Mentioned in Section 2.3</td></tr><tr><td>Amplifier</td><td>Homemade</td><td></td><td>Voltage amplification of the signal from the signal generator to the electrodes. Mentioned in Section 2.3</td></tr><tr><td>Power supply</td><td>Matrix&amp;nbsp;</td><td>MPS-3005 LK-3&amp;nbsp;</td><td>Power supply to the sputtering machine. Mentioned in Section 1.2.2.3</td></tr><tr><td>Transcranial magnetic stimulation</td><td>Magstim, Spring Gardens, UK</td><td>Rapid 2</td><td>Magnetic stimulation of neuronal culture. Mentioned in Sections 3.1, 3.3, and 3.4</td></tr><tr><td>Epoxy</td><td>Cognis</td><td>Versamid 140</td><td>Casting of homemade coils. Mentioned in Section 3.4</td></tr><tr><td>Epoxy</td><td>Shell</td><td>EPON 815&amp;nbsp;</td><td>Casting of homemade coils. Mentioned in Section 3.4</td></tr><tr><td>Platinum wires 0.005&#039;&#039; thick; A-M Systems,&amp;nbsp;</td><td>&amp;nbsp;Carlsborg WA</td><td>&amp;nbsp;767000</td><td>Electric stimulation of neuronal cultures. Mentioned in Section 2.1</td></tr><tr><td>Circular magnetic coil</td><td>Homemade</td><td></td><td>Magnetic stimulation of neuronal culture. Mentioned in Section 3.3</td></tr><tr><td>WaveXpress SW</td><td>B&amp;amp;K Precision&amp;nbsp;</td><td></td><td>Waveform editing software. Mentioned in Section 2.1.32</td></tr><tr><td>Xion Ultra 897</td><td>Andor</td><td></td><td>Sensitive EMCCD camera. Mentioned in Section 2.4.4</td></tr></tbody></table>

external excitation of neurons using electric and magnetic fields in one- and two-dimensional cultures

当它的膜电位超过某个阈值的神经元就会触发动作电位。在大脑中的典型的活性，这发生如化学投入到它的突触的结果。然而，神经细胞也可以通过强加的电场激发。尤其是，最近的临床应用程序激活由外部产生电场的神经元。因此，感兴趣的研究神经元如何响应外场是什么原因导致的动作电位。幸运的是，外部电场的精确和受控应用是可能的被切下，解离和在培养物中生长的神经元的胚胎细胞。这使得在一个高度可重复的系统，这些问题的调查。 在本文中的一些用于外部电场对神经元培养物控制的应用程序的技术进行了综述。这些网络可以是一维的，在LINEA 即图案化ř形式或允许在基板的整个平面成长，因此二维的。此外，激发可通过电场直接应用通过浸入流体（浴电极）或通过使用磁脉冲的远程创建诱导电场的电极产生。

<html>提出的协议允许神经元培养物的容易构图。当它与我们为刺激开发了几种方法相结合，它能够使一些内在的神经元属性，如时值和Rheobase 5的测量，比较健康和疾病的神经元27的特性，找到最佳的方法来刺激文化之间的函数关系它们的结构和更多的新方法。一些例子，在未来的数字呈现。 <p class="jove_content" fo:keep-together.within-page="1"...

Watch this Scientific Journal Video about External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures at JoVE.com

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

神经元培养是通过其对单个神经元或神经元群体的影响来研究新兴脑刺激技术的良好模型。这里提出的是通过直接由浴电极产生的电场或由时变磁场诱导的电场刺激图案化神经元培养物的不同方法。

使用电动和一维和二维文化磁场神经元的外部激励

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

external-excitation-neurons-using-electric-magnetic-fields-one-two

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13.2K 次观看.  The Salk Institute for Biological Studies. 神经元培养是通过其对单个神经元或神经元群体的影响来研究新兴脑刺激技术的良好模型。这里提出的是通过直接由浴电极产生的电场或由时变磁场诱导的电场刺激图案化神经元培养物的不同方法。 

视频: External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

电场控制的二维和三维环境中的神经祖细胞定向迁移

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

科研

生物工程

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

一个的趋电含量为神经前体细胞迁移的动力学分析外部施加直流电场

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

神经科学

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. 
We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

螺旋神经节神经元外植体培养及电多电极阵列

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

时间依赖性增加对微电极阵列刺激神经元细胞培养物的网络响应

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.

硅纳米线与光学刺激研究细胞内和细胞间电耦合

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These ...

Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.

In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system.

微流体设备中的电场诱导神经前体细胞分化

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

用于刺激生物组织的电动和磁场设备

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

External Excitation of One-Dimensional Patterned Neuronal Cultures

Source:Stern, S., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54357">External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures.</a> J. Vis. Exp. (2017)
This video demonstrates a technique for stimulating line-patterned neuronal cultures with a uniform, unidirectional electric field.

一维模式神经元培养物的外部激发

1. Imaging of spontaneous or evoked activity in neuronal cultures with fluorescent dyes.

	Prepare a solution of 50 &#181;g calcium sensitive fluorescent ...

JoVE Encyclopedia of Experiments

Neurophysiology

Stimulation and Modulation Techniques

Stimulation of Patterned Neuronal Cultures by a Magnetic Field

Source: Stern, S., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/54357">External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures.</a> J. Vis. Exp. (2017)
This video demonstrates a method for stimulating neuronal cultures grown in a patterned circular ring on a coverslip, using a time-varying magnetic field.

磁场刺激模式化神经元培养物

1. Preparation of coverslips for 1D cultures.

	Clean glass coverslips by immersion in a base piranha solution consisting of 75 mL DDW, 25 mL 25% ammonia ...

Building Three-Dimensional Neuronal Networks Coupled to Micro-Electrode Arrays

Source: Tedesco, M., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/53080">Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model.</a> J. Vis. Exp. (2015)
This video demonstrates the development of a 3D neuronal culture coupled to a planar microelectrode array with hippocampal neurons. The isolated neurons form a 2D network on the microelectrode array&#39;s active area and adhere to glass microbeads in a multi-well plate with a membrane insert. Upon transferring these neuron-coated microbeads to the electrode, they organize into an interconnected 3D neural network.