我们要感谢宫原一郎多次有帮助的讨论和见解。 MA和PG承认NSERC的资金。

以下详细说明的所有步骤均由麦吉尔大学动物护理委员会批准，并符合加拿大动物护理委员会的指导原则。 使用微流控器件进行神经元培养的标准化:器件装配<ol><li>为所需实验选择合适的微流体装置。为了连接相同群体中的神经元，使用Neuro Devices（ 图1 ），并连接不同人群中的神经元使用共培养装置（ 图6 ）。 </li><li>清洁并准备所需数量的无菌盖玻片或玻璃底盘。为了在塑料表面上获得最佳效果，请使用35毫米的菜肴，玻璃上使用25毫米的盖玻片或35毫米的玻璃底盘。根据成像系统选择玻璃厚度，例如0.15 mm。 </li><li>用0.5-1mL的100μg/ mL PDL将盘或盖玻片涂覆2小时或室温过夜ATURE。 注意:如果需要，协议可以暂停，并在第二天恢复。还可以用硼酸盐缓冲液稀释的PDL，聚-L-赖氨酸（PLL），层粘连蛋白或任何其他细胞粘附分子来涂覆菜肴。 </li><li>用水洗涤两次（不要使用磷酸盐缓冲盐水（PBS），因为盐水晶体可能堵塞通道），除去所有的液体，让其在无菌环境如生物安全柜中干燥5-10分钟或直到表面完全干燥。 注意:小心确保盖玻片绝对干燥，因为任何剩余的液体会干扰微流体系统的粘附。 </li><li>将无菌环境（生物安全柜）内的图案面向紫外线的微流体装置放置10分钟。在生物安全柜10工作时，请务必遵循无菌程序。 </li><li>使用镊子放置一个微型流体装置，图案面朝下，与干净的盖子接触IP /皿。使用镊子轻轻按下设备，使其粘附在玻璃上。 注意:当与光线对准时，粘附区域的透明度将是可见的。确保所有角落都与玻璃接触。对所有微流体设备进行此操作。参见图1a 。 </li><li> 用培养基填充单个种群装置，将移液管移至通道，加入补充无血清B-27（体积比1:50）和500μg/ mL青霉素/链霉素/谷氨酰胺的50μL完全细胞培养基称为NBM）到右上部孔，然后向对角线放置另外50μL。对所有设备执行此操作，确保介质在井之间流动。接下来，向剩余的2口孔中加入50μL培养基。参见图2a 。 <ol><li>要用介质填充多种群体装置，将移液管指向通道并加入30μL完整的NBM到右侧井，参见图6a 。对所有设备执行此操作，确保介质在井之间流动。接下来，向其余4口井中加入50μL培养基。 </li></ol></li><li>将设备放置在带有高压灭菌水（湿室）的开放盘的较大板上，并置于培养箱（37℃，5％CO 2和95％湿度）中1-2小时，同时准备细胞培养。参见图2b 。 </li></ol>微流控系统中的电镀神经元<ol><li>遵循参考文献中概述的方案。 8 ，从Sprague Dawley大鼠胚胎（性别）获得解离的海马或皮质神经元。 </li><li>在NBM中以1-2万个神经元/ mL的浓度重悬胚胎神经元。使用血细胞计数器检查显微镜中的细胞浓度，并参考文献8 。调整细胞浓度g到所需的细胞密度。为了增加每个通道获得单个海马轴突的机会，每个装置平板10,000个神经元。要在同一个通道中有多个轴突，每个设备可以放置60,000个神经元。 注意:这些数字根据所使用的神经元类型而有所不同。 </li><li>从微流体装置中取出培养基，而不排空孔。每个留下约5μL。 </li><li> 为了在单个群体装置中平板培养细胞，向右下方加入50μLNBM。在这一点上，介质自身流动以填充另一个较低的井。将20μL浓缩液溶液加入微流体装置的右上方， 如图1b所示 。 <ol><li>为了在多种群装置中平板培养细胞，在图6a的每个右孔中加入20μL浓缩细胞溶液。 </li></ol></li><li>检查显微镜下是否有细胞在室内并将装置置于培养箱中15-30分钟以促进细胞附着于基质。 </li><li>检查显微镜，如果室内有足够的细胞。如果需要更多，请重复步骤2.4和2.5。 </li><li>将50μLNBM添加到单个群体装置的2个顶部孔中，并将20μLNBM加入到与多个群体装置中注入细胞的相同的孔中。介质稍微突出，形成一个阳性弯月面，给予一个松饼顶部的孔。再次参见图2a 。 </li><li>保持细胞在37°C，5％CO 2和95％湿度。 </li></ol> 3.保持神经元文化<ol><li>从细胞中取出NBM（用移液管大约30μL），并在引入设备后的第一天（即步骤2后1 d）应用新的预热NBM。 </li><li>如果每个通道中有足够的介质，请每2天检查一次。如果松饼top是低的，只是添加更多的中等水平的井。 </li><li>培养细胞在除去微流体装置之前至少7天。细胞可在这些装置中存活数周。在对样品进行实验前1-2天取出设备。 </li></ol>微流控器件的去除<ol><li>在去除微流体装置之前1-2天，向每个样品盘预加热至37℃的2毫升NBM，淹没室，并将装置保持在培养箱中。 </li><li>使用无菌镊子和一个尖端从盖玻片上移除微流体装置，留下神经元的图案化构型。使用尖端将盖玻片固定到位，镊子将孔的左下角扣住设备的边缘。巧妙地施加扭力，用镊子将装置抬起，以便从盖玻片上剥落。参见图2c - 2d 。 </li><li>每2-3天换一次如果NBM直到样品用于实验。 </li><li>在对样品进行重新布线实验之前，请先通过检查显微镜间的间隙来确认单个群体装置通道中的神经突和多重群体装置中的神经元群体是否分离，以确保没有连接神经元群体的细丝。 </li></ol> 5.准备PDL涂层珠<ol><li>加入2×50μL将在水（1:500）中稀释的4,10或20微米聚苯乙烯珠滴至1毫升的PDL（100微克/毫升）。在室温下放置至少2小时。 注意:协议可以暂停，并在第二天恢复。 </li><li>将溶液以8,820 xg离心1分钟。仔细清除上清液，不会干扰容器底部积聚的珠子。 </li><li>用1 mL无菌10 mM HEPES pH 8.4溶液洗涤珠子两次。 </li><li>将PDL包被的珠粒重悬在200mL的10mM HEPES pH8.4中解。 </li></ol>准备微量移液器<ol><li>使用水平电极牵引器从玻璃毛细管（1mm内径，1.5mm外径）准备移液器。调整设置，使拉出的微量移液器的外尖端〜2-5μm。在拉动之前，确保玻璃管是干净的。 </li><li>将移液器固定到玻璃载玻片上进行存放，并确保尖端不易接触滑块表面，因为尖端易碎。在室温下存放在有盖的容器中，以防灰尘。在同一天使用移液器。 </li></ol> PDL珠粒与神经元的粘附<ol><li>将在步骤5中制备的40-60μL的PDL包被的珠粒加入到步骤4中制备的细胞培养基中。将移液管尖端移动到盖玻片上微弱可见的神经元上，并加入珠（参见图3 ）。 </li><li>将样品送回培养箱1小时以促进sy的形成咔嗒声接触8,9 。 </li><li>孵育后，通过用预热NBM轻轻洗涤培养物去除任何未附着的珠粒。 </li></ol> 8.准备生理盐水溶液（室温实验） <ol><li>通过组合参考文献7,8中列出的成分制备生理盐水溶液。这是为了调节孵化器外的细胞环境。 </li><li>验证渗透压和pH值，如参考文献7,8 所示 。 </li><li>在进行实验的同时，用O 2连续注入溶液以使pH波动最小化。 </li><li>加热至室温。 </li><li> 通过将塑料管的一端（可选尺寸）插入O 2引入的生理溶液中并固定另一个e来设置灌注系统nd插入插入样品架中的针。将管道和溶液放置在样品上方（ 见图4 ）。 <ol><li>将管与针断开并将其连接到注射器。使用注射器施加压力并抽出液体，填充管子。用滚筒夹将其密封并重新连接针头。 </li></ol></li></ol>珠粒微操作<ol><li>将样品安装在实验装置中，使得可以通过安装在显微操纵器中的两个微量移液器从上方访问细胞，并且通过倒置光学显微镜的40X相位物镜（数值孔径0.6）光学下方访问细胞。在此配置中，将CCD摄像头安装在显微镜侧面的图像捕获。通过塑料管将每个移液器连接到1 mL注射器。在此步骤中，用生理盐水溶液（1-2 mL）代替NBM（ 见图4 ）。 </li><li>在实验期间用0.5-1mL / min的速率在步骤8中制备的生理盐水不断地灌注细胞。 </li><li>选择未附加到视野中的神经元的PDL珠。通过聚焦到珠上，然后直到微量移液管，将珠与微量移液管尖对齐。通过显微镜监测，将尖端尽可能靠近珠子。 </li><li>使用连接到移液器的1 mL注射器吸取珠来施加负压。在整个实验过程中保持负压。 </li></ol>拉神经元<ol><li>选择在视野中连接到神经元的PDL珠，并使用如步骤9.3-9.4中所述的吸力将其附着到第二个微量吸管。 </li><li>通过缓慢（〜0.5μm/ min）拉动PDL珠 - 神经元复合物，将微操纵器或样品台移动1μm，并暂停5分钟以允许神经突起始。 </li><li>重复步骤10.2两次。 注意:前3个＃181; m必须被拉得很慢，以保证实验成功，这超过了95％的时间。 </li><li>通过缓慢（〜0.5μm/ min）拉动PDL珠 - 神经元复合物，将微操纵器或样品台移动2μm，暂停5 min，以允许神经突伸长。 </li><li>在第一个5μm的成功引发和神经突延伸后，以毫米级距离以20μm/ min拉动神经突。 注意:拉伸可以连续地或者以不同的速率逐步进行。 见图5b -5c 。 </li></ol>连接神经元<ol><li>选择一个富含神经突的区域，并降低PDL珠珠神经突复合体，使其与物理接触。使用其他珠子来测量盖玻片表面以上的尖端高度。 见图5d 。 </li><li>离开PDL珠珠神经突复合体与目标神经突接触，同时操纵第二个微点pette。将第二个移液管放在新形成的约20μm左右的神经突的顶部，第二个PDL珠形成第一个珠。使用第二个PDL珠将新的神经突细丝推向目标细胞。 </li><li>将两个珠子保持在适当位置至少1小时。用显微镜16验证不存在局部肿胀，与珠接触的神经突的增厚。 </li><li>在此期间，使用灌注将样品的介质从生理盐水慢慢地改变为预热的CO 2均衡的NBM。 </li><li>通过释放吸力从第二吸管释放珠子。如果新的神经突仍然附着，也释放第一个珠子。 见图5e 。 </li><li>轻轻取出生理盐水，用NBM（〜2 mL）取代。 </li><li>仔细将样品放回培养箱中，以加强神经元连接，以便将来进行实验。此连接稳定&gt; 24小时7 </sup&gt;。 </li></ol> 12. 通过全细胞配对贴片记录验证新连接的功能<ol><li>按照参考文献7,18,19。组装电生理设置。 </li><li>参考文献7 。以准备前和突触后电极。 </li><li>收集膜片钳数据，再次参考18,19。 </li><li>将结果与自然发生的信号7进行比较，以确定连接类型。 </li></ol>

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作者Margaret H Magdesian是Ananda Devices的首席执行官，负责本文中使用的仪器。

使用标准显微操作和创新的微流体装置，开发了一种新技术，可以在很大距离上快速启动，延长和精确连接新的功能性神经元电路。移液管显微操作是大多数神经科学实验室4,13中的常用工具。实现可重复和可靠结果的真正挑战是通过开发微流体装置，以微米精度组织细胞培养，在实验期间（可能在几周内）将健康精确定位的神经元培养物标准化。高质量的细胞培养是数据验证的基石。这有助于更容易和更快速的显微成像和标准化文化和结果。微流体装置设计用于体外培养细胞，其体内具有相似的组织。单个人口设备启用长轴突的生长和容易识别轴突，神经突和神经细胞。在微流体室中铺板的细胞数确定神经元密度。因此，通过每个器件电镀较少的细胞，容易识别靠近通道的单个神经元。一个单个神经元的轴突和多个树突在一个通道内生长。树突状体比轴突6,17生长至少5倍，体外 2-3周后，通道中的生长通常限于200μm，而快速生长的轴突可以达到超过2毫米17 。因此，在将PDL珠添加到样品后，相对容易估计珠粒是否主要粘附于轴突或轴突和树突（ 图5b - 5f ）。拉动附着于短于200μm的神经突的PDL珠的新轴突和树突的机会更大，while在拉长PDA珠附着于超过500μm的神经突时，有更大的可能性拉扯新的轴突。多重群体装置对于健康分离群体的可重复生长有用数周。这种通道系统可用于研究多达4种不同的细胞类型或将不同细胞进行不同的处理。 此外，受控和可重复的细胞培养环境为细胞分析和操作提供了理想的框架。细胞在盘上的精确定位有助于识别感兴趣的区域，以及通过这些感兴趣的区域的取向和导航，最终允许对神经突起始位点进行前所未有的控制。受控的细胞分布使得更容易将新形成的连接物可视化，并且在孵化之后的几天内用显微镜找到新的连接更快。此外，细胞的重复配置使得能够容易和精确地定位化学线索，例如PDL涂覆的珠粒，在索马里，树突或轴突上8 。总之，由于在所有菜肴中再现的标准细胞组织，在微流体装置中生长的细胞的成像和分析更快。此外，器件由生物相容，透明和可移动的材料制成，使得能够在器件内的所有可见波长和细胞存活下成像数周。细胞测定的微型化也有助于以更少的细胞收集更多的数据。微流体装置的低体积消耗减少了每个实验所需的细胞数量并增加了实验效能。例如，不用在显微镜上花几个小时在盘中搜索约1或2个孤立的轴突，而是可以使用单个种群装置，以便每个细胞样本立即获得超过100个孤立的轴突。微流体装置提供可靠和可控的小型化细胞培养环境有利于对稀有样品的分析进行多次测试。 该技术的潜在变化涉及将珠直接附着到微量移液管。在当前的协议中，描述了一种用于通过抽吸将胎圈附接到尖端的方法，但是胎圈也可以胶合到尖端。如果需要尖端和珠之间的高度稳定的连接，则粘胶是更好的选择，例如如果使用移液管作为传感器进行力测量，或者PDL和神经突之间的粘附性调查是主要的实验目标。在另一个静脉中，抽吸是有利的，因为它允许放置后的珠粒释放而不断裂神经突 - 珠复合体，从而能够并行地进行多个连接。吸力提供了高通量重新布线实验的手段，相对于其他操作技术的改进，如原子力显微镜（AFM）/ sup&gt; ， 16 。这种方法的两种修改允许通过简单地保持与枝晶接触的珠子来选择神经突起始位点，从而形成突触。然而，如步骤7所述孵育几个珠子的策略在操作阶段节省了时间，并且如果在实验中不需要对起始部位进行精确控制，则被推荐。 未来的研究可能寻求解决各种工具限制，即温度控制和样品可及性。细胞膜的机械性能可以在不同的温度下显着变化27 。理想情况下，所有实验应在37℃下在神经元培养基和适当条件（正确的CO 2压力和湿度对照）下进行。然而，不可能使用闭孔培养箱，因为对于显微操作器需要从顶部进入样品，从底部用于显微镜和从侧面移动舞台。因此，使用室温灌注。类似地，由于设置仅具有足够的空间来容纳2个微操纵器，并且需要将近1小时以建立单个连接，所以可以执行的实验的数量是有限制的。这个问题可以通过一次拉动多个珠子的操纵器来解决。该协议的另一个潜在的改进是从样品表面注册珠移液管复合体的高度。不能做到这一点可能会导致不精确的时候，把第二个珠子放在诱导的神经突上来固定它。珠粒在富含神经突的区域的新神经突的顶部下降，直到新的神经突和盘上的那些突起位于相同的焦平面上。新的神经突从不在珠子和盘子之间，但总是在细胞物质的垫子和珠子之间，因此压缩减小。另外，在显微镜下观察新的神经突10分钟，以评估在珠附近是否形成神经突局部肿胀。如参考文献16所述，溶胀的存在表示神经突变性。然而，由于对轴突施加不同程度的压力可能导致生理变化16 ，未来的研究可能集中于通过将反射器固定到其上来适应移液管探针，并通过AFM方法监测与样品表面垂直的位移。最后，也许这个协议最大的挑战是测试被操纵的连接的功能。最直接，可靠和成熟的技术是配对的全细胞膜片钳记录。然而，常规配对膜片钳记录的成功率非常低（&lt;25％） 18 。全细胞膜片钳有几个缺点，包括设置时间长，实验次数有限，录制时间有限（〜30）min），配对膜片钳记录的实验产量低，测定后细胞死亡。由于这些技术挑战，显微操作后全细胞膜片钳配对记录的实验产量非常低。需要更好的平台和技术来更精确地刺激，记录和比较自然和显微操作连接中的神经元活动。 自神经解剖学的早期以来，已经知道轴突生长中的紧张的重要性 - 将其称为被动伸展20 。在早期胚胎发育过程中，神经突迁移很短距离以达到目标。随着大多数细胞分裂和复制，轴突受到连续的力量的伸长，并将其长度调整到胚胎生长20,21 。几个组试图通过向neu施加化学线索和/或机械张力来测试神经突生长的极限（参见参考文献22 ）。在这些报告23,24,25,26中，以及在生理生长过程中，在附着于基质的同时牵引神经突。与我们的设置的主要区别是，在本技术中，新的神经突只有两个粘附触点;在基部，神经突附着于神经元，尖端附着于珠粒。在伸长期间，新神经突的组分具有以最有效的方式分散以适应拉力的自由。更重要的是，该协议描述了如何再现这些实验，而不会引起神经突破裂或退化，这是基于先前关于突触接触形成的研究8以及轴突对压力16的阻力，显示如何使用微量和纳米工具用适当的力量持续拉动神经突。这里描述的神经突延伸率（1.2mm / h）比来自周围神经系统（0.02至0.04mm / h）的最快生长的轴突的体内速率快30-60倍。当与体外相同的神经元类型相比时，这里描述的轴突延伸率比其他作者在早期发展阶段（0.1mm / h） 4描述的生长速度快7.5倍。已经发现不同类型的神经元以不同的固有速率延伸轴突，其变化几倍29 。另外，中枢神经系统轴突通常在体外靶向神经支配体外失去高轴突生长率29和3 d的体外培养 6 。因此，目前的轴突延伸技术应该用不同的神经元类型进行测试，以更好地理解e神经突延伸的限制。 移液管显微操作和微流体装置是证明创建新的功能神经突和可控地定位或（重新）线神经元网络的技术。该平台是系统，标准化测量的理想选择。它将重现性和体内控制引入到神经元复杂网络的实验中。实现的扩展速率比毫米级距离快20微米/分钟，建立功能连接。这些结果意外地显示，轴突的延伸的内在能力，包括其细胞骨架成分的内在能力比以前认为的10,11,12快得多。这种提出的机械方法绕过慢化学策略，因此代表了治疗发展恢复损伤后神经元连通性的范式转变并用于人造神经网络的微神经工程用于其体外对照研究。这些结果也对再生医学和神经工程方法产生重大影响，直接影响旨在在创伤后或神经退行性疾病中重新连接神经元回路的疗法。该平台打开了获取神经元通信，信号调制以及增长和再生的数据的大门。这是机械再生CNS的一种新方法，类似的技术可能会在损伤后恢复功能。此外，该技术可用于创建系统地设计的神经元网络作为用于药物发现和目标验证的新型生物测定平台。它是强大的脑机接口的直接接线的前身。

成人中枢神经系统（CNS）的损伤可能导致永久性残疾，由于多种机制限制轴突再生长1 。受伤后，许多CNS轴突不形成新的增长锥，不能建立有效的再生反应2 。此外，围绕CNS损伤的损伤和瘢痕组织显着抑制轴突生长1,2,3 。目前用于促进损伤后CNS再生的疗法主要集中在增强受损神经元的内在生长潜力，并掩盖与髓磷脂碎片和胶质瘢痕1,3相关的轴突延伸抑制剂。尽管如此，将长轴突再生到远处目标并形成适当的功能性突触的能力仍然受到严重限制4 ， </sup&gt; 5,6,7 。 在目前的工作中，微珠，移液管显微操作和微流体装置用于在长距离上快速启动，延长和精确连接新的功能性神经元电路。以前的工作已经显示聚-D-赖氨酸包被的珠粒（PDL珠）诱导膜粘附，然后突触囊泡复合物的聚集和功能突触前boutons的形成8 。还显示当PDL珠被突触前分化后机械地拉开时，突触蛋白簇跟随珠，引发新的神经突9 。以下程序利用这一事实以及使用聚二甲基硅氧烷（PDMS）微流体装置将大鼠的胚胎海马神经元培养成盖玻片上的有组织区域以将精神重新连接神经元电路。 这些PDMS微流体装置是无毒的，光学透明的，由通过微通道系统连接的两个室组成。一旦组装在盖玻片上，每个装置用作引导神经元生长的模具，并且在体外将精确模式维持健康的神经元培养超过4周。 在这里，提出了一个框架来研究新神经突的扩展和功能的限制。新的功能神经突被创建并定位成可控地（重新）连线的神经元网络。实现的扩展速率比毫米级距离快20微米/分钟，建立功能连接。这些结果意外地显示出这些神经突伸长的内在能力比以前想象的要快得多。这种提出的机械方法绕过了缓慢的化学策略，并实现了与特定目标的控制连接。钍是技术开创新的疗法的新途径，以恢复损伤后的神经元连通性的新疗法。它还使得神经元网络的操纵和重新布线能够在体外研究神经元信号处理和神经元功能的基本方面。

<table><tbody><tr><td>Co-culture devices</td><td>Ananda Devices</td><td></td><td>Commercially available at http://www.anandadevices.com</td></tr><tr><td>Neuro devices</td><td>Ananda Devices</td><td></td><td>Commercially available at http://www.anandadevices.com</td></tr><tr><td>No. 1 Glass Coverslip 25 mm Round</td><td>Warner Instruments</td><td>64-0705</td><td></td></tr><tr><td>35 mm Glass Bottom Dishes #0, Uncoated, Gamma-Irradiated</td><td>MatTex Incorporation</td><td>P35G-0-20-C</td><td></td></tr><tr><td>35 mm cell culture dish, Non-Pyrogenic, Sterile</td><td>Corning Inc</td><td>430165</td><td></td></tr><tr><td>95 mm x 15 mm Petri Dish, Slippable Lid, Sterile Polystyrene</td><td>Fisherbrand</td><td>FB0875714G</td><td></td></tr><tr><td>50 mL Centrifuge tubes with printed graduations and flat caps</td><td>VWR</td><td>89039-656</td><td></td></tr><tr><td>15 mL Polypropylene Conical Tube, 17 x 120 mm style, Non Pyrogenic, Sterile</td><td>Falcon</td><td>352097</td><td></td></tr><tr><td>Neurobasal Medium</td><td>Life Technologies</td><td>21103-049</td><td>Extracellular solution</td></tr><tr><td>B-27 Supplement (50X), serum free</td><td>B-27 Supplement (50X), serum free</td><td>17504044</td><td>Extracellular solution</td></tr><tr><td>Pennicilin, Streptomyocin, Glutamine</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>11995-065</td><td>Extracellular solution</td></tr><tr><td>200 &amp;mu; L Pipettors</td><td>VWR</td><td>89079-458</td><td></td></tr><tr><td>2 - 20 &amp;mu;L Pipettors</td><td>Aerosol Resistant Tips</td><td>2149P</td><td></td></tr><tr><td>BD Falcon 3mL Transfer Pipettes [Non-sterile]</td><td>BD Falcon</td><td>357524</td><td></td></tr><tr><td>Glucose</td><td>Gibco</td><td>15023-021</td><td>Extracellular solution</td></tr><tr><td>HEPES</td><td>Sigma</td><td>7365-45-9</td><td>Extracellular solution/Beads</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>7647-14-5</td><td>Extracellular solution</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>7447-40-7</td><td>Extracellular solution</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>10043-52-4</td><td>Extracellular solution</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>7786-30-3</td><td>Extracellular solution</td></tr><tr><td>#5 Dumont Dumostar Tweezers 11 cm</td><td>World Precision Instruments</td><td>500233</td><td></td></tr><tr><td>Dissection tools</td><td>Braun, Aesculap</td><td></td><td></td></tr><tr><td>Poly-D-lysine Hydrobromide</td><td>Sigma-Aldrich</td><td>P6407</td><td></td></tr><tr><td>Micro particles based on polystyrene, 10 &amp;mu;m</td><td>Sigma-Aldrich</td><td>72986</td><td></td></tr><tr><td>Borosilicate tubes</td><td>King Precision Glass, Inc.</td><td>14696-2</td><td></td></tr><tr><td>Horizontal Pipette Puller</td><td>Sutter Instruments</td><td>Brown-Flaming P-97</td><td></td></tr><tr><td>Micromanipulators, PCS-5000 Series</td><td>SD Instruments</td><td>MC7600R</td><td></td></tr><tr><td>1 mL Syringe</td><td>BD Luer-Lok</td><td>309628</td><td></td></tr><tr><td>Inverted Microscope</td><td>Olympus&amp;nbsp;</td><td>IX71</td><td></td></tr><tr><td>Objective</td><td>Olympus</td><td>UIS2, LUCPLFLN 40X</td><td></td></tr><tr><td>CCD Camera</td><td>Photometrics</td><td>Cascade II: 512</td><td></td></tr><tr><td>Leibovitz&#039;s (1x) L-15 Medium</td><td>Life Technologies</td><td>11415-064</td><td>Rat Dissection</td></tr><tr><td>Typsin-EDTA (0.05%), Phenol red</td><td>Life Technologies</td><td>25300054</td><td>Rat Dissection</td></tr><tr><td>DMEM (1x) Dulbecco&#039;s Modified Eagle Medium [+4.5 g/L D-Glucose, + L-Glutamine, + 110 mg/L Sodium Pyruvate]</td><td>Life Technologies</td><td>11995-065</td><td>Rat Dissection</td></tr><tr><td>HBSS (1x) Hank&#039;s Balanced Salt Solution [- Calcium Chloride, - Magnesium Chloride, - Magnesium Sulfate]</td><td>Life Technologies</td><td>14170-112</td><td>Rat Dissection</td></tr></tbody></table>

rewiring neuronal circuits: a new method for fast neurite extension and functional neuronal connection

脑和脊髓损伤可能导致永久性残疾和死亡，因为仍然不可能长距离再生神经元，并准确地将其与适当的目标重新连接。这里描述了一种快速启动，延长和精确连接长距离功能神经元电路的过程。实现的延伸率达到1.2mm / h以上，比来自周围神经系统（0.02至0.04mm / h）的生长最快的轴突的体内速率快30-60倍，比先前报道的速度快28倍和10倍早期发育阶段的神经元类型4 。首先，分离的大鼠海马神经元群体在微流体装置中生长2-3周以精确定位细胞，使得易于显微操作和实验重现性。接下来，将涂覆有聚-D-赖氨酸（PDL）的珠子放置在神经突上以形成粘合剂作用和移液管显微操作用于移动所得珠粒神经突复合体。当珠移动时，它拉出一个可以在数百微米上延伸的新神经突，并在不到1小时内与靶细胞功能连接。该过程使实验重现性和易于操作，同时绕过较慢的化学策略来诱导神经突生长。这里提出的初步测量表明，神经元生长速度远远超过生理因素。结合这些创新，可以以前所未有的程度来精确地建立文化中的神经元网络。这是一种新颖的方法，为神经元网络中的信号传输和通信开辟了大量的信息和洞察力，也是探索神经元生长极限的操场。潜在的应用和实验是广泛的，直接涉及旨在重新连接神经元的治疗创伤后或神经退行性疾病中的电路。

将胚胎大鼠海马神经元在微流体装置中培养以使细胞，PDL珠和显微操纵体的精确定位。第一步是将微流体装置适当地组装在玻璃盖玻片或盘子上。必须使微流体装置良好地附着到基底上以避免细胞离开腔室并移动到应密封的装置的部分（ 图1a ）。为了保持健康的文化数周，重要的是通过每2-3天检查细胞培养基并保留培养基的阳性弯液面来防止培养基蒸发（ 图2a ）。通过将细胞和开放的水保持在更大的板内（ 图2b ）也可以避免中等蒸发。可以随时移除微流体装置。为了获得最佳结果，应将NBM添加到cell-de副系统在设备移除前至少1天。当器官被去除时，细胞应力最小化，因为神经元在理想温度和pH下与培养基接触。当微流体装置从盘缓慢剥离（ 图2c 和2d ）细胞将保留在图案化位置（ 图3和图5a ）。 使用两种类型的微流体装置:神经装置和共培养装置。第一个可以轻松识别轴突，树突和细胞体。神经瘤保留在顶部神经腔室中，而轴突和树突沿着微流体通道（ 图5a ）朝向轴突腔室生长。 建议以10:1的比例将PDL珠添加到细胞中，大多数珠子加入到细胞中在1小时孵育后，通过用NBM洗涤细胞一次来除去培养物（ 图3 ）。随着PDL珠粘附到神经突，PDL珠珠神经突复合体被拉伸并可以在很大的距离上延伸。新形成的神经突可以精确地连接到神经突或毫米之外（ 图5b -5e ）。在低于1μm/ min的速度下将前3μm拉伸新神经突的成功率&gt; 95％（n = 206）。将新神经突连接到另一个细胞的成功率为70％（n = 30）。连接在前18小时非常脆弱，主要是因为新的神经突是由10μm的PDL珠填充的直径小于1μm的细丝。如果盘在接触的第一分钟内快速移动，则所得到的介质湍流可能引起胎圈滚动，从而粘附将丧失。但是，如果样本不是shaken，在30分钟内，珠子附着在盘子上的神经突，新的连接保持至少48小时。有关设置的示意图，请参见图4 。 培养物上的PDL珠位置也可用于容易地识别连接的神经突的位置（ 图6d-6e ）。在引发之后，通过微操纵器拾起的新的神经突，第二个非粘附的PDL珠被开始，延伸和连接，用于在引发的神经突和第二个神经元之间产生第二个粘附部位。第二个PDL珠放置在新神经突的顶部，压缩它，使得新的神经突刚刚接触第二个神经元11 。这将促进与第二神经元群体的粘附（ 图5f ）。 多人群装置启动在同一道菜中有4个孤立的神经元种群的生长。每个神经元群体被限制在与其他神经元群体分隔开的距离为100或200微米的4×7毫米矩形（ 图6a ）。健康的神经元可以在设备内长达数周。通常，在移除装置后，神经元群体保持隔离至48小时。在这48小时之后，神经元趋向于向相邻的神经元群体生长并形成自然的连接。在连接两个孤立的群体之前，应该证实，通过用显微镜检查整个差距来确定群体确实是真正的分离，以确定两个神经元群体之间没有联系（ 图6b ）。 连接后，将样品孵育24小时，并进行电整体细胞配对膜片钳记录，以研究是否新的用于连接两个孤立的神经元群体的神经突是功能性的，能够传输电信号。选择位于距诱发神经突起始位点半径小于100μm的神经元记录突触前动作电位（PAPs）。该神经元被认为是突触前细胞。从位于距离显微操作连接的半径小于100μm的间隙的另一侧的人口2的神经元记录突触后兴奋性或抑制活性（ 图7a- 7c ）。分析来自机械诱导连接的记录，并与来自天然连接的神经元群体和非连接群体的记录进行比较（ 图7 ）。从天然连接的神经元和通过显微操作记录的PAP记录的电响应显着更高，并且与突触前时间相关活动（ 图7 ）。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55697/55697fig1.jpg" /> 图1: 使用微流控装置的神经元培养物的标准化 。 （ a ）设备组装:当微流体装置在干燥的表面上正确组装时，所有的室都是可见的。 （ b ）细胞电镀:将右上方的细胞平板化，细胞向左移动。 （ c ）细胞密度:电镀后，检查细胞浓度是否足够。 （ d ）培养1 d后，海马神经元很好地附着在微通道上，开始形成神经突。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55697/55697fig1large.jpg" target="_blank">请点击这里查看大图</a><html>呃这个数字版本。 <img alt="图2" class="xfigimg" src="/files/ftp_upload/55697/55697fig2.jpg" /> 图2 :维持健康的神经元培养数周7 。 （ a ）每2-3天添加培养基，并在微流体室的上部孔中保持阳性弯月面，因此细胞将具有恒定的营养供应。 （ b ）用含有水的盘将细胞放入更大的板中以减少中等蒸发。 （ c ）使用无菌尖端和镊子（ d ）轻松剥离微型设备。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55697/55697fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> /。。。。。。。。。。。。 图3 :移液管沉积珠在培养物中的位置。一旦微流体装置被去除，神经元就可以在盖玻片上看到。当沉积珠子时，定位移液管尖端使其位于细胞的中心。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55697/55697fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/55697/55697fig4.jpg" /> 图4: 典型的神经元牵引装置示意图。样品搁置在（压电致动）阶段，可以从上方通过2个安装在显微操纵器中的微量移液器进行访问，并通过塑料管连接到1 mL注射器。样品通过连接到发送的CCD相机的物镜从下方光学地进行访问s图像到CPU。入口管将氧化生理盐水溶液供给到其下方的样品，并且连接到注射器的出口管允许在溢出的情况下提取溶液。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55697/55697fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图5" class="xfigimg" src="/files/ftp_upload/55697/55697fig5.jpg" /> 图5 :PDL-珠粒与神经元的粘附和移液管的微操作 。 （ a ）除去微流体室之后，神经元依然以图案形式组织，从而容易地鉴定出神经突起和神经突。 （ b ）粘附于神经突的PDL珠的微操作使神经突起始，延伸（ c ）和连接（ d ），随后从移液管（ e ）释放PDL珠。 （ f ）接触两个分离的神经元群体（底部箭头）后，使用第二个PDL珠和移液管显微操纵器（顶部箭头）建立第二个粘附点，第一个接触点相隔数百微米，并保证功能连接。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55697/55697fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图6" class="xfigimg" src="/files/ftp_upload/55697/55697fig6.jpg" /> 图6 。使用多种群体设备和微操作连接两个孤立人口的新神经元的启动，伸长和连接 7 。 （ a ）该装置分离4个神经元群体在每个100或200微米的3个间隙之间。 （ b ）移除装置后，选择一个缺口并证明没有神经突连接2个个体。 （ c ）在光学显微镜中应该可见的实验装置的示意图表示两个微量移液管的位置和PDL包被的珠子的存在。 （ d ）通过向移液管施加负压，用移液管吸头牵引附着于一个神经元群的PDL珠，从而引发新的神经突。通过保持移液管中的负压，可以拉伸PDL珠 - 神经突复合体（绿色），延长神经突。 （ e ）移液管显微操作指导新的神经突延伸在间隙上，并与新的神经元群体形成连接。为了确保新神经突向第二群体的粘附，将PDL珠（红色）定位为与第二移液管位于两个延伸的神经突和神经的顶部人口总数<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55697/55697fig6large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图7" class="xfigimg" src="/files/ftp_upload/55697/55697fig7.jpg" /> 图7: 新诱导的，细长的和连接的神经元可以在两个孤立的神经元群体之间传递信息 。分离的神经元群体通过PDMS微器件中的100μm间隙进行培养分离。当两个群体通过机械操纵（ a ）连接时，从群体1的神经元和群体二（在间隙的另一侧）的神经元，以整个细胞构型进行配对的膜片钳记录，允许自然地互连间隙（ b ）或由维护点保持不连接缩小差距（ c ）。每个条件（ df ）显示了成对记录的代表性痕迹。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55697/55697fig7large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection at JoVE.com

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

该程序描述了如何使用固定在引导神经突伸长的微量移液管上的聚-D-赖氨酸包被的珠快速启动，延伸和连接在微流体室中组织的神经突。

重建神经元电路:快速神经延伸和功能性神经元连接的新方法

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

rewiring-neuronal-circuits-new-method-for-fast-neurite-extension

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

8.7K 次观看.  McGill University. 该程序描述了如何使用固定在引导神经突伸长的微量移液管上的聚-D-赖氨酸包被的珠快速启动，延伸和连接在微流体室中组织的神经突。 

视频: Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

评定神经连接的形成在共培养体系的光遗传学方法

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

科研

发育生物学

Perspectives on Neuroscience

As usual, the main cause for progress in natural sciences are methodological developments. Of course, conceptual developments follow and go in parallel, but I think the, the tremendous expansion of the field of neurosciences was triggered by a number of methodological breakthroughs at the lowest level. These were the progresses in the domain of molecular biology, genetic engineering, which provided model systems for the study of development for the study of certain diseases.These techniques also provided extremely valuable tools for the biochemical and molecular identification of neurons. We learned about a degree of diversification of morphologically seemingly identical neurons that are endowed with different molecular machineries. All this we owe to the development of molecular biology.A second important factor I think was the development of non-invasive imaging technologies. cause this allowed us to transfer a large number of concepts that have been developed in animal experimentation to investigations in human subjects. So this is the functional magnetic resonance imaging pository emission tomography.Also the introduction of the magneto ence holography, which allowed for much better spatial resolution for the analysis of electric activity in the brain led to a substantial expansion in the field. Then I think a non negligible factor was the availability, increasing availability of of massive computer power because as neurobiology started to apply multi-site recording techniques, which I consider essential for the analysis of neuron network properties, the data became increasingly complex and it became increasingly impossible to just evaluate the data by looking at them as in the classical times when I started in the field, there were no computers. We had to do cross correlation analysis by cutting long strips of paper and moving them over the floor.So the availability of of computing power has greatly advanced at least systems physiology. I think it had a major impact on these developments. And then last but not least, the availability of these computer facilities allowed us also to treat properties of neuronal networks at a theoretical level.The dynamics exhibited by neuronal networks are so complex that only modeling studies and theoretical approaches put us in a position to formulate educated hypothesis. Also, for the search of neuronal codes, we cannot proceed without these search tools. So I think altogether these methodological developments were responsible for the great expansion of the neurosciences, which I think is unprecedented in any Scientific discipline.As far as my own Research is concerned, there are two parts in my biography. Initially I studied developmental processes in the brain. I was particularly interested in developmental processes that are shaped by experience postnatally.And the rationale for this approach was that I felt that the organization of the mature brain is so complex that we better study the developmental principles and try to make sense out of them and try to extrapolate what these developmental processes will eventually lead to. But then an accidental finding that actually happened in this very laboratory where we are sitting changed my life because I discovered inadvertently that neurons in the primary visual cortex of kittens that were awake actually sitting on the table behind me watching a drifting grating, got entrained into highly synchronous oscillatory activity that I could not attribute neither to degrading to the stimulus nor to the main switch in Germany are 50 cycles because it was an oscillation in the 40 hertz range. And so for the first time I saw that spatially distributed neurons could coordinate their activity with very high precision in the millisecond range over fairly large distances.And this triggered the idea that maybe time is used as a coding space for the coordination of distributed neuronal responses. And I found this option so interesting that I decided to neglect the so much beloved field of developmental neurobiology and invest most of our efforts into the further analysis of this phenomenon. And this is now nearly 15 years ago or more.And since then we spent most of our time with multi-site recordings and the search for spatial temporal patents in distributed neural networks because it is our firm belief that information is not only encoded in the discharge rate of neurons as is classically assumed and confirmed, but that additional information is encoded in the precise temporary relations among the individual discharges of neurons whereby zero phase lake correlations. So precise coincidence is probably only the tip of the iceberg. But an important tip because synchronous activity is particularly interesting in neuronal networks because it propagates very easily synchronizing responses is a means to enhance the saliency of neuronal responses with great temporal position.It can be done on a spike by spike basis. And the initial hypothesis was that this synchronization is used at peripheral stages of sensory processing in order to establish relations between features that are processed by spatially distributed neurons. Since then, many more labs have engaged in search for the synchronization phenomena.It started I think in Europe with colleagues like RI who applied EEG techniques to assess synchronous oscillatory activity in the brain and relate these activities to cognitive functions. And since then the the role of oscillations and synchrony has been discussed in a very controversial way. Initial doubts that this phenomenon might exist, it all have now been countered I think, and we are now all engaged in the search for functional consequences.And there is a whole plethora of hypothesis of what that could mean. Does the accumulation Of this vast amount of data that we are confronted with nowadays and that we have very great difficulties to cope with reflect true progress. It certainly does reflect progress because we get to know more and more facts and details about the organization of neurons, neuron networks and the brain as a whole.And this is a necessary consequence of the classical analytical approaches that are applied in the natural sciences. One isolates a subsystem and then describes it in ever greater detail. But this is only one half of the scientific endeavor.What we also need is the inductive strategy. This the synthesis of all those data. And what we are now confronted with is that we have a huge amount of words.We have a tremendous vocabulary that is difficult to remember, but we are lacking the syntax to put those words together into meaningful sentences in many domains. This is the case in particular in systems neuroscience. Therefore we need, I think more theory because conditions have become so complex that near verbal descriptions are probably no longer sufficient to grasp or to accomplish this, this synthetic process.We need formal descriptions, we need analysis tools that allow us to generate models and this requires very powerful computations. And then we need of course synthetic thinkers. That's probably the the most important ingredient in the moment.People who can bundle a large amount of details into meaningful hypothesis or concepts. Now one can of course foster this process by providing the appropriate environment and worldwide one sees efforts to provide such environments by generating institutions that are usually called Institute for Computational Neuroscience or Institute for Theoretical Neuroscience. For the time being, many of these places are still existing in two great in isolation and are not sufficiently linked to experimental institutions.Ideally one would have, one would like to see a very close cooperation between theoreticians and experimentalists, as has been so fruitful in physics where theoretical physicists and experimental physicists very closely collaborate freely, exchange data ideas and do this without jealousy, which is still not the case in our field. But I think biology is now approaching a threshold where it is mature enough to support theoretical approaches in a meaningful way. And here in Frankfurt, we have founded the Frankfurt Institute of Advanced Studies exactly for that purpose because I experienced that physicists and mathematicians, theoreticians in general when embedded in the experimental institute, they tend to to be marginalized.They don't have enough colleagues to talk to because biologists usually are not trained in their language. And so the idea was let's create an institute in which several model systems, biological model systems that have in common to be multi-component systems that are tightly coupled like the moon system or the brain are studied by theoreticians, hoping that these theoreticians might discover common principles in these complex self-organizing systems and that they can share analytical tools, can share tools for model building. And I think it worked out because within about a year this institute grew from zero to 120 scientists.And the special structure of this institute, which is not surprising to colleagues in America, but this is quite exceptional in Europe, is that this institute is funded exclusively by donors private funds. It is not a university institute, it is linked to the university in Frankfurt, but it is finance in a different way for the simple reason that it was impossible to install such an interdisciplinary enterprise within the conventional faculties because what happens there is truly I the disciplinary, it crosses faculties and they seem to be realizable only by having a erect and an own building and gather people who come from very different disciplines under the same roof. Even though those Terms have been taken from physical systems initially, they still grasp some of the characteristics of of of the brain because the brain clearly is a self-organizing system.There's no conductor in the brain to coordinate all the distributed processes. The brain develops in a self organizing way through interaction between genes and environment. There is no instructor, there is no blueprint, a priori.And finally the term applies very well to what we observe in complex systems all the time, namely that complex systems tend to exhibit properties that cannot be described or inferred from the properties of the components. And the brain is a particularly nice example for this fact, which we know from all complex systems there's nothing special about the brain. Something that is special about the brain is that we have on the one side material processes, neuron interactions that can be described from a third person perspective as any other process in our environment.And that emergence from these neuronal interactions are mental phenomena, psychological phenomena such as emotions, thoughts, decisions. So there are realities which I would like to address as social realities which emerge or come into being by the interaction between brains that communicate with each other, mirror each other, ascribe properties to each other, could replace brains by persons, doesn't really matter because what makes the person is finally the brain in its in its bodily setting and due to these interactions phenomena emerge that are to be taken as real phenomena, they are realities, but they have to be described in a different language as the phenomena that bring them forth that are at their origin, namely neuronal interactions. And therefore I think that this is a classical case of emergence where something new is generated by a complex system, nobody would ever be able to infer from the knowledge, complete knowledge of neurons, and even from complete knowledge of neuron networks, that they are capable of developing value systems when they interact With each other.I think the main Problem in our efforts to repair brains, apart from the fact that neurons do not easily regenerate in the central nervous system, they don't like to extend processes once they are severed for reasons that we do not know. Maybe there was no Evo evolutionary pressure on the development of such mechanisms because brain damage that really leads to cell death in in a large scale was usually not compatible with survival. The great problem in restoring neuron networks that have been damaged is that it is difficult to recapitulate the developmental process.The mature brain is characterized by an extremely sophisticated circuitry that has developed step by step in a complex self-organizing process, guided by the dialogue between the genes and the increasingly more differentiated cellular environment that changes throughout development. And this process is a essentially bottom up process. Peripheral structures mature first, then they go through critical periods and the connectivity becomes fixed so that the, the higher stages can rely upon what has already been built and then they organize themselves as a function of what's already there.And this process goes on in human beings until age 20 and then the circuitry crystallizes and then we have to live with the brains that we have got at that point of time. There's further modification by learning and neuroplasticity, but this is limited. Now if one were to replace neurons at higher levels of processing, for example, by injection of stem cells or so these neurons we know already, they can differentiate, they the precursors can become neurons, but the problem is will they find the partners with which they have to interact?Will the information still be there That was available during development that guided the axon growth, that guided the formation of specific connections. And we know that to, to a large extent, this information is no longer available because the genes that one needs in order to express the necessary marker molecules, they get switched off once development comes to an end. And what is probably necessary is that one not only supplies cells that can differentiate into neurons and of course also ggl cells, but that one also switches on or helps the organism to switch on the genes that have been active during the developmental process that lead to the expression of all the molecular machinery that is required to tell the cells with whom they should connect, where they should connect.And what of course cannot be reinstalled easily is all the idiosyncratic wiring and the adjustment of synaptic game that is due to learning processes. The newly integrated neurons will of course have no souvenir, no recollection of the experience that has been imprinted into the morphology and the structure and the function of neurons that have disappeared. So there are certain limits to the ability to restore.And as we realize in the moment, there are certain places in the brain where this seems to pose less of a problem because nature has foreseen lifelong regeneration of neurons and apparently kept the machinery alive that is needed in order to promote this ongoing regeneration and insertion of neurons as in hippocampus and in the olfactory bulb. But the other structures where this is probably causing great problems, the first replacements that have been attempted and have had certain success even though it was not satisfactory yet, where the modulatory systems where topological specificity is not that much a problem. These systems are functioning more like endocrine or paracrine systems that liberate certain transmitters that are required in order to keep a certain tous of excitability.These systems are more easily replaceable because specificity is less of a problem, but the future will show how far we get there. Certainly very promising approaches in reinstalling oxon outgrowth in several spinal cords by inhibiting factors that inhibit outgrowth in the adult and by activating mechanisms that come to action during development and promote outgrowth. There are encouraging successes in this field, but one should also consider very seriously the option of neuronal prosthetics to record neuronal activity from places where it is still available and then through mechanical devices and computer interfaces activate actors that can translate this activity into action for the motor system.Or conversely, on the sensory side, cochlear implants already work admirably well. There are now attempts to develop retinal implants in order to reinstall rudimentary visual functions in people who have retinal degeneration. There's also a very promising option to insert light gated iron channels into retinal cells so that cells that are normally not light sensitive, which are the ganglion cells for example, or the bipolar cells become light sensitive so that one could in principle reins store some function that is lost when photoreceptors degenerate by simply transferring light sensitivity to second or third order neurons.This is already possible in animal experimentation. It might be another promising avenue to pursue. I think Europe has a very strong tradition in the development of concepts end of 19th century helm those seminal figures who have provided the grounds for later experimental neurobiology to some extent.They also are the grand grandfathers of a substantial number of systems physiologists who have been active in Europe. I think there is a good tradition in systems neuroscience in Europe has always been in the times when, when linear systems theory was still at its peak cybernetics, as it was called Europe, had a few very strong exponents such as rehad for example. Then the, there was something like, not iconic turn, but the molecular turn in Europe as well as anywhere else, molecular biology took over and replaced many of the systems oriented approaches.Partly because initially it had been cheaper than to work with whole animals, partly because non-invasive technology was not available and one couldn't do certain things in animals, which can now be done in human subjects. But I think there's a strong renaissance in systems physiology even though in Europe we have to fight a hard battle against animal protectionists concerning primary research, this is a real critical issue. The labs that can, they still get the license to work with primates become less and less and those who still have the license.This is the case with us where we have, we are exposed very much to public critique, but still systems neuroscience is having a renaissance. And I think there are good reasons for that because we will need it more and more in order to make sense out of the plethora of data that are collected by the properties of neurons and small circuits. And these data can only be integrated if we have concepts derived from systems neuroscience on the nature of the neuronal code, on the cooperation of areas in the brain, on global coordination of processes, on distributed processing.All those questions are heatedly debated in the moment and we just need more experiments along those lines. And I think Europe is not in a bad position to do this. Israel as well, one should not forget the contribution, also conceptual contribution of our colleagues in Israel, which is substantial.

展望神经

生物学

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

诱导多能干细胞的快速神经元分化有关微电极阵列测量网络活动

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

工程神经前体宫内移植,用于Vivo神经结构研究

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

遗传学

An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic dysfunction occur in various neurodegenerative diseases and age-related degeneration. Investigation of the mechanisms that regulate neurite outgrowth would not only shed valuable light on brain developmental processes but also on such neurological disorders. Due to the low transfection efficiency, it is currently challenging to study the effect of a specific protein on neurite outgrowth in primary mammalian neurons. Here, we describe a simple method for the investigation of neurite outgrowth by the co-transfection of primary rat cortical neurons with EGFP and a protein of interest (POI). This method allows the identification of POI transfected neurons through the EGFP signal, and thus the effect of the POI on neurite outgrowth can be determined precisely. This EGFP-based assay provides a convenient approach for the investigation of pathways regulating neurite outgrowth.

In this report, we describe a simple protocol for studying neurite outgrowth in embryonic rat cortical neurons by co-transfecting with EGFP and the protein of interest.

增强的绿色荧光蛋白测定，用于研究原神经元的神经质生长

Neurite outgrowth is a fundamental event in the formation of the neural circuits during nervous system development. Severe neurite damage and synaptic ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

神经元共培养与单细胞精度的制备

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

神经科学

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 ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

生物工程

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire of dynamical motifs, which underlie most diverse brain functions, can emerge out of a fixed topological and modular organization of brain circuits. Compared to in vivo studies of neuronal circuits which present intrinsic experimental difficulties, in vitro preparations offer a much larger possibility to manipulate and probe the structural, dynamical and chemical properties of experimental neuronal systems. This work describes an in vitro experimental methodology which allows growing of modular networks composed by spatially distinct, functionally interconnected neuronal assemblies. The protocol allows controlling the two-dimensional (2D) architecture of the neuronal network at different levels of topological complexity.
A desired network patterning can be achieved both on regular cover slips and substrate embedded micro electrode arrays. Micromachined structures are embossed on a silicon wafer and used to create biocompatible polymeric stencils, which incorporate the negative features of the desired network architecture. The stencils are placed on the culturing substrates during the surface coating procedure with a molecular layer for promoting cellular adhesion. After removal of the stencils, neurons are plated and they spontaneously redirected to the coated areas. By decreasing the inter-compartment distance, it is possible to obtain either isolated or interconnected neuronal circuits. To promote cell survival, cells are co-cultured with a supporting neuronal network which is located at the periphery of the culture dish. Electrophysiological and optical recordings of the activity of modular networks obtained respectively by using substrate embedded micro electrode arrays and calcium imaging are presented. While each module shows spontaneous global synchronizations, the occurrence of inter-module synchronization is regulated by the density of connection among the circuits.

This manuscript describes a protocol to grow in vitro modular networks consisting of spatially confined, functionally inter-connected neuronal circuits. A polymeric mask is used to pattern a protein layer to promote cellular adhesion over the culturing substrate. Plated neurons grow on coated areas establishing spontaneous connections and exhibiting electrophysiological activity.

设计，表面处理，电镀细胞和神经元的模块化网络功能组成跨接电路的培养

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire ...

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

解剖学启发的三维微组织工程神经网络用于神经系统重建，调制和建模

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...