作者感谢乔治卡烈尼在制定限制结构和多特的技术支持。的Ornella LoBrutto对稿件的彻底的修改。研究导致这些结果已收到的资金来自欧盟的7 个框架计划（ICT-FET FP7 / 2007-2013年FET年轻探险家计划）下拨款协议284772脑弓（www.brainbowproject.eu）。

实验方案是按照DL一千九百九十二分之一百一十六和热那亚（普罗特大学的指导方针获得了欧洲动物保护立法（2010年/ 63 / EU），由卫生意大利外交部。N. 13130​​，五月2011）。所作的所有努力减少动物项目的数量，并尽量减少他们的痛苦。 1.准备材料，并支持<ol><li>构建模具由CNC（计算机数控）磨粉机的方式来构建PDMS（聚二甲基硅氧烷）的约束。这种模具被实现在聚碳酸酯与聚四氟乙烯中央筒（PTFE）。这种材料允许容易提取荫罩一旦与PDMS已硬化。 注：该模具的设计已经通过计算机辅助-设计（CAD）中进行，然后输送到数控铣床。 </li><li>准备PDMS弹性体。 PDMS是一种有机聚合物。它是由两个元件，固化剂和聚合物。通过1:10的体积比混合使用它们，一个部分固化剂，9份聚合物。把两种组分在培养皿，摇并插入真空室中的混合物10分钟，以消除气泡。 3克PDMS是足够的一个制约因素。 </li><li>构建PDMS约束。将PDMS材料进入成型机，并把它放入烤箱30分钟，在120℃。将PDMS约束具有理想缸筒用的22.0和3.0毫米，分别外部和内部的直径和650微米的高度的形状。 </li><li>电镀的前一天，夫妇由镊子的装置在立体显微镜下对掩模的微电极阵列（MEAs）中的有效区域和消毒的PDMS掩模在烘箱中在120℃。 </li><li>消毒玻璃微珠（40±2微米的标称直径; 42.3±1.1微米的认证平均直径）在70％乙醇处理2小时在锥形瓶中并旋转EVERY 30分钟，揭露所有的微珠。 </li><li>除去从管形瓶中的乙醇溶液和冲洗微珠两次用无菌水。 </li><li>调节所述MEA面积由PDMS掩模在0.05微克/毫升分隔（直径等于3.0毫米），24微升聚-D-赖氨酸层粘连蛋白和混合溶液的中心部分。 </li><li>外套与粘附蛋白，层粘连蛋白和聚-D-赖氨酸的微珠在0.05微克/毫升和离开他们过夜，在培养箱中在37℃，以获得稳定和持久的神经元网络。 </li><li>既从MEA的玻璃表面，并从该玻璃微珠除去粘附因子。抽吸用移液器涂布溶液的约95％，应用的无菌水的小体积 - 24μl-在MEA的表面。抽吸再次超过95％的水，让层流罩1小时下在MEA干镀细胞之前。 注意：在微珠的情况下，而不是，吸出涂层溶液并进行第一次洗涤用无菌水，而第二个与基础培养基和其补充如B27，以调节所述的玻璃表面，其中神经元将被电镀。不要让微珠干。其保留在悬浮液中的小瓶内培养基中。 </li><li>分发，通过吸管-200μl-，处理微珠的悬浮液（32000）的方式到多孔板与膜插入（孔径0.4微米），他们将自组装形成均匀的一层。 </li><li>填充膜的每个孔与0.5培养基中，并把它放在孵化器（T = 37.0℃，CO 2 = 5％），直到神经元是准备镀（3.2）。 </li></ol> 2.解剖胚胎和组织的解离<ol><li>房子成年雌性大鼠（200-250克）中的正则明暗时间表的恒定温度（22±1℃）和相对湿度（50％）（光7 AM-7 PM）中的动物设施。确保食物和水都是免费的。 </li><li>后用3％异氟醚18天发展（E18）麻醉怀孕的雌性大鼠。然后，通过颈椎脱位牺牲动物。 </li><li>从每个大鼠胚胎中取出海马并将其放置在冰冷的汉克的无钙 ，镁平衡盐溶液2+。在第18天发育海马和皮层组织的非常柔软，不需要被切成小块。进一步的细节可以发现18,19。 </li><li>离解在含有DNA酶的0.05％为18-20分钟胰蛋白酶/汉克溶液0.125％的组织，在37℃。 </li><li>除去用巴氏吸管将上清液溶液并用5分钟加入培养基，用10％胎牛血清（FBS）停止酶消化。 </li><li>用FBS除去介质并用与它的补充，1％L-谷氨酰胺，庆大霉素10μg/ ml的培养基洗一次。用巴斯德点子除去再次介质ETTE并再次用生长培养基少量（500微升）与它的补充，1％L-谷氨酰胺，庆大霉素10μg/ ml的填充它。 </li><li>具有窄巴斯德吸管机械解离组织粒料直到细胞的乳状悬浮液是显而易见的。它没有必要离心细胞悬浮液。 </li><li>稀释小体积的细胞悬浮液与生长培养基，得到2.0 ml的终体积。计数与血球室所得到的细胞浓度。为了得到600-700个细胞/微升的期望的细胞浓度为5：稀释该浓度为1。 </li></ol> 3.细胞电镀<ol><li>板的细胞以约2000个/ mm 2的密度到MEA的有源区域，由PDMS约束定义来创建2D神经网络。 注意：各海马包含约5×10 5个细胞中，（1×10 6，用于单个胚胎）。解剖6胚胎获得的6×10的总量6细胞在2ml，和3000个细胞/微升的估计浓度。为了获得所需的细胞浓度和板大约600-700个细胞/微升5：稀释该浓度为1。如果在MEA面积由PDMS约束分隔为约7.065毫米2，并铺板细胞总数600个细胞/微升×24微升= 14400细胞，2038个/ mm 2的最终密度。 </li><li>放置在MEA器件与湿润的 CO 2气氛（5％）培养箱，在37℃。 </li><li>分发160微升的悬浮液用600-700个细胞/微升（约100,000个细胞）的细胞浓度到微珠单层放置在多孔板内的表面，以完成三维培养的结构的初步步骤。把多孔板在培养箱加湿的 CO 2气氛（5％），在37℃。 </li></ol> 4. 3D神经元网络建设<ol><li> 6-8小时的电镀后，从多孔板非常仔细转移悬浮液（微珠与神经元的）分隔由PDMS约束通过使用移液管为约30-40微升的体积设置的区域内。每次传输后，等待半分钟左右，以使微球自组装的六边形结构紧凑。 </li><li>一旦所有的层被沉积并自发地聚集，填充用一大滴的约300微升培养基由PDMS约束限定的区域的顶部。 </li><li>把耦合到MEA的3D结构中的保育箱（T = 37.0℃，CO 2 = 5％）48小时加入生长培养基培养物的最终体积（约1毫升）与它的补充之前。 注意：考虑的珠子上的多孔板与膜插入件（30,000）和珠上的单一层上的MEA装置（6000）的数量的总数。将所得的三维结构是由5层英里的crobeads和细胞。考虑到所述三维神经网络不完美几何，所得到的三维结构可以由5-8层。 </li><li>在天铺板后，小心地添加培养基（约900微升）在MEA环内的最终体积。保持在湿润的 CO 2气氛（5％）的3D培养，在37℃下进行4-5周。更换每周一次培养基的一半。 </li></ol> 5.共聚焦显微镜采集<ol><li>固定的生物样品，以在支架的显微镜阶段。设置激光（氩，496-555纳米），扫描速度（400赫兹），在此获得PMT的图像，所述增益和偏移（-0.3％）为每个图像捕获正确的带宽发射滤波器，并为了以避免饱和，并提高信​​噪比。结果部分报告用于获取图4的图像值。 </li><li>对于此次收购的 Z -stack序列，冷杉圣选择，顶部和样品的底层的z位置的值，然后选择步长，使采集。 </li></ol>

<ol>
	<li>Van Pelt, J., Corner, M. A., Wolters, P. S., Rutten, W. L. C., Ramakers, G. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+stability+and+developmental+changes+in+spontaneous+network+burst+firing+patterns+in+dissociated+rat+cerebral+cortex+cell+cultures+on+multi-electrode+arrays.">Long-term stability and developmental changes in spontaneous network burst firing patterns in dissociated rat cerebral cortex cell cultures on multi-electrode arrays.</a> Neurosci. Lett. 361, 86-89 (2004).</li><li>Rolston, J. D., Wagenaar, D. A., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precisely+timed+spatiotemporal+patterns+of+neural+activity+in+dissociated+cortical+cultures.">Precisely timed spatiotemporal patterns of neural activity in dissociated cortical cultures.</a> Neuroscienc. 148, 294-303 (2007).</li><li>Frey, U., Egert, U., Heer, F., Hafizovic, S., Hierlemann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microelectronic+system+for+high-resolution+mapping+of+extracellular+electric+fields+applied+to+brain+slices.">Microelectronic system for high-resolution mapping of extracellular electric fields applied to brain slices.</a> Biosens. Bioelectron. 24, 2191-2198 (2009).</li><li>Macis, E., Tedesco, M., Massobrio, P., Raiteri, R., Martinoia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+microdrop+delivery+system+for+neuronal+network+patterning+on+microelectrode+arrays.">An automated microdrop delivery system for neuronal network patterning on microelectrode arrays.</a> J. Neurosci. Meth. 161, 88-95 (2007).</li><li>Kanagasabapathi, T. T., Ciliberti, D., Martinoia, S., Wadman, W. J., Decre, M. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+compartment+neurofluidic+system+for+electrophysiological+measurements+in+physically+segregated+and+functionally+connected+neuronal+cell+culture.">Dual compartment neurofluidic system for electrophysiological measurements in physically segregated and functionally connected neuronal cell culture.</a> Front. Neuroeng. 4, (2011).</li><li>Kanagasabapathi, T. T., 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=Functional+connectivity+and+dynamics+of+cortical-thalamic+networks+co-cultured+in+a+dual+compartment+device.">Functional connectivity and dynamics of cortical-thalamic networks co-cultured in a dual compartment device.</a> J. Neural. Eng. 9, (2012).</li><li>Cullen, D. K., Wolf, J. A., Vernekar, V., Vukasinovic, J., LaPlaca, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+tissue+engineering+and+biohybridized+microsystems+for+neurobiological+investigation+in+vitro+(Part+1).">Neural tissue engineering and biohybridized microsystems for neurobiological investigation in vitro (Part 1).</a> Crit. Rev. Biomed. Eng. 39, 201-240 (2011).</li><li>Wagenaar, D. A., Madhavan, R., Pine, J., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+Bursting+in+Cortical+Cultures+with+Closed-Loop+Multi-Electrode+Stimulation.">Controlling Bursting in Cortical Cultures with Closed-Loop Multi-Electrode Stimulation.</a> J. Neurosci. 25, 680-688 (2005).</li><li>Shany, B., Vago, R., Baranes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+primary+hippocampal+neuronal+tissue+on+an+aragonite+crystalline+biomatrix.">Growth of primary hippocampal neuronal tissue on an aragonite crystalline biomatrix.</a> Tiss. Eng. 11, 585-596 (2005).</li><li>Schmidt, C. E., Leach, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+tissue+engineering:+strategies+for+repair+and+regeneration.">Neural tissue engineering: strategies for repair and regeneration.</a> Ann. Rev. Biomed. Eng. 5, 293-347 (2003).</li><li>Ma, W., 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=CNS+stem+and+progenitor+cell+differentiation+into+functional+neuronal+circuits+in+three-dimensional+collagen+gels.">CNS stem and progenitor cell differentiation into functional neuronal circuits in three-dimensional collagen gels.</a> Exp. Neurol. 190, 276-288 (2004).</li><li>Baranes, D., 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=Interconnected+network+of+ganglion-like+neural+cell+spheres+formed+on+hydrozoan+skeleton.">Interconnected network of ganglion-like neural cell spheres formed on hydrozoan skeleton.</a> Tissue En. 13, 473-482 (2007).</li><li>Lee, J., Cuddihy, M. J., Kotov, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+culture+matrices:+state+of+the+art.">Three-dimensional cell culture matrices: state of the art.</a> Tissue engineering. Part B, Review. 14, 61-86 (2008).</li><li>Schuz, A., Palm, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Density+of+neurons+and+synapses+in+the+cerebral+cortex+of+the+mouse.">Density of neurons and synapses in the cerebral cortex of the mouse.</a> J. Comp. Neurol. 286, 442-455 (1989).</li><li>Pautot, S., Wyart, C., Isacoff, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colloid-guided+assembly+of+oriented+3D+neuronal+networks.">Colloid-guided assembly of oriented 3D neuronal networks.</a> Nat. Method. 5, 735-740 (2008).</li><li>Frega, M., Tedesco, M., Massobrio, P., Pesce, M., Martinoia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+dynamics+of+3D+engineered+neuronal+cultures:+a+new+experimental+model+for+in-vitro+electrophysiology.">Network dynamics of 3D engineered neuronal cultures: a new experimental model for in-vitro electrophysiology.</a> Sci. Rep. 4, (2014).</li><li>Tang-Schomer, M. D., 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=Bioengineered+functional+brain-like+cortical+tissue.">Bioengineered functional brain-like cortical tissue.</a> Proc Natl Acad Sci U S. 111, 13811-13816 (2014).</li><li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cell. , (1998).</li><li>Hales, C. M., Rolston, J. D., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+culture,+record+and+stimulate+neuronal+networks+on+Micro-electrode+arrays+(MEAs).">How to culture, record and stimulate neuronal networks on Micro-electrode arrays (MEAs).</a> J. Vis. Exp. (39), (2010).</li><li>Jayakumar, R., Prabaharan, M., Nair, S. V., Tamura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+chitin+and+chitosan+nanofibers+in+biomedical+applications.">Novel chitin and chitosan nanofibers in biomedical applications.</a> Biotech. Adv. 28, 142-150 (2010).</li><li>Crompton, K. E., 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=Polylysine-functionalised+thermoresponsive+chitosan+hydrogel+for+neural+tissue+engineering.">Polylysine-functionalised thermoresponsive chitosan hydrogel for neural tissue engineering.</a> Biomaterial. 28, 441-449 (2007).</li><li>Frega, 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=3D+engineered+neural+networks+coupled+to+Micro-Electrode+Arrays:+Development+of+an+innovative+in-vitro+experimental+model+for+neurophysiological+studies.">3D engineered neural networks coupled to Micro-Electrode Arrays: Development of an innovative in-vitro experimental model for neurophysiological studies.</a> , 957-960 (2013).</li></ol>

The authors declare that they have no competing financial interests.

在这项工作中，一个新的实验体外平台由三维设计的耦合到多边环境为网络电已提交的神经元培养物。使用微珠为支架，以允许沿 z轴的神经炎生长已针对与平面MEA被集成。以这种方式，所得到的微系统导致有效和可靠的体外三维模型来研究所述紧急电动力学16。 MEA记录的建立 MEA设备包括一个文化室的底物嵌入式微电极能够?...

体外二维（2D）神经耦合到微电极阵列（MEAs）的网络正像空通过研究神经元动力学和底层连接之间的相互作用黄金标准实验模型。在发展过程中，神经细胞再造的复杂网络，其显示效果也很好definedspatio，temporalpatternsof活性1,2（ 即阵阵，网络阵阵，随机扣球活动）。多边环境协定记录的电生理活动从许多网站（从几十到几千微电极），使表达的动态在网络层进行详细的调查。另外，使用解离的培养物的不可能性使得设计工程-网络。这是比较容易用这种方式来理解和所记录的电生理活动之间的功能关系样细胞密度3，模块化4,5的程度，异构神经流行的存在的网络组织的参数ulations 6等。然而，所有在体外研究上解离的培养细胞是基于二维的神经元网络。这种方法导致过分简单化相对于所述体内 （本质三维，三维）系统：（i）在二维模型中，胞体和生长锥变平及轴突-树突生长不能散布在所有方向7。 （二）2D 体外网络表现出刻板通过爆破涉及大部分的网络8的神经元的活动为主的电动力学。 最近，不同的解决方案已经开发了允许在体外构建3D解离的神经元网络。在共同的理念在于创造一个支架，其中神经元可以在一个三维的方式成长。这样的支架可以实现与聚合物凝胶和固体多孔基质9-13通过利用聚合物的机械性能，有可能以嵌入内采取这些细胞Ë结构定义神经球11的3D文化的统一块。这种方法的主要特征是神经球9,12的刚性的机械性能。然而，这些材料具有有限的孔隙率，并且他们不保证基质内细胞迁移。为了克服这个缺点，一种可能的解决方案包括在切片矩阵变成&#39;单元&#39;的模块。不幸的是，颗粒的大小和形状的多样性会妨碍包装成规则的层状结构。 7，Cullen和同事设计内的生物活性细胞外基质为基础的支架由神经元和/或星形胶质细胞的神经元的三维结构。这种工程化神经组织允许在体外的调查研究和操作中的3D微环境的神经生物学反应。这个模型包括神经元和神经胶质细胞在整个细胞外基质（ECM）和/或水凝胶支架（500-600微米厚）分布的。在这种合作ndition，最佳细胞存活率（大于90％），发现通过在约3750的最终密度电镀细胞- 5000 细胞 / mm3。必须指出的是，这样的密度值小于一个在体内条件下，其中小鼠大脑皮质的细胞密度为约90000个细胞/ mm 3的14低得多。为了克服这个限制Pautot和同事15实现其中的细胞密度和网络连接被控制在体内条件类似于同时使网络的实时成像一个三维的体外系统 。实际上，该方法是基于解离培养的神经元是能够生长在硅胶上微珠的概念。这些珠子提供生长表面的神经元胞体坚持和他们arborizations成长，成熟，扩展和定义突触联系到其他神经元足够大。这种方法利用了单分散珠自发组装性能FORM 3D分层含不同层，不同的珠子神经元之间的约束连接神经元的不同的子集六角形阵列。用这种方法所达到的细胞密度为约75000个/ mm 3。 最近，我们已适应Pautot的方法来多边环境16：所获得的结果表明，该三维电生理学活性呈现比所述一个二维网络表示活动更宽剧目。 3D成熟的文化表现出增强的动态，其中两个网络的突发和随机秒杀活动并存。同样，汤-Schomer和同事17实现可维持原代皮层体外培养数月丝蛋白质基多孔支架，并通过一个钨电极的装置记录的电生理活性。 在这项工作中，实验步骤建立联接到多边环境三维神经元网络进行说明。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td>0.05 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P6407</td>
			<td>0.05 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>25050-014</td>
			<td align="left">0.125% diluted 1:2 in HBSS w\o CA++, MG++</td>
		</tr>
		<tr>
			<td>DNAase</td>
			<td>Sigma-Aldrich</td>
			<td>D5025</td>
			<td>0.05% diluted&#160; in Hanks solution</td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco Invitrogen</td>
			<td>21103049</td>
			<td>culture medium</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco Invitrogen</td>
			<td>17504044</td>
			<td>2% medium supplent</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F-2442</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Glutamax-I&#160;</td>
			<td>Gibco</td>
			<td>35050038</td>
			<td>0.5 mM</td>
		</tr>
		<tr>
			<td>gentamicin</td>
			<td>Sigma-Aldrich</td>
			<td>G.1272</td>
			<td>5mg/liter</td>
		</tr>
		<tr>
			<td>Poly-Dimethyl-Siloxane (PDMS)</td>
			<td>Corning Sigma</td>
			<td>481939</td>
			<td>curing agent and the polymer</td>
		</tr>
		<tr>
			<td>Micro-Electrode Arrays</td>
			<td>Multi Channel Systems (MCS)</td>
			<td>60MEA200/30-Ti-pr</td>
			<td>MEA with: Electrode grid 8x8; Electrode spacing and diameter 200 and 30 &#181;m, respectively; plastic ring without thread</td>
		</tr>
		<tr>
			<td>Microbead</td>
			<td>Distrilab-Duke Scientific</td>
			<td>9040</td>
			<td>&#160;1gr Glass Part.Size Stds 40 &#181;m</td>
		</tr>
		<tr>
			<td>Transwell</td>
			<td>Costar Sigma</td>
			<td>CLS 3413</td>
			<td>multiwell plates with membrane insert (6.5 mm diameter porous 0.4 &#181;m)</td>
		</tr>
		<tr>
			<td>HBSS w\o Ca++ ,Mg++&#160;</td>
			<td>Gibco Invitrogen</td>
			<td>14175-052</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks Buffer Solution&#160;</td>
			<td>Sigma&#160;</td>
			<td>H8264</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon</td>
			<td>Sigma</td>
			<td>430935-5G</td>
			<td>polytetrafluoroethylene</td>
		</tr>
		<tr>
			<td>Rat</td>
			<td>Sprague Dawley</td>
			<td>Wistar Rat</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscopy Upright</td>
			<td>Leica</td>
			<td>TCS SP5 AOBS</td>
			<td></td>
		</tr>
		<tr>
			<td>20.0x0.50 WATER objective</td>
			<td>Leica</td>
			<td>Leica HCX APO L U-V-I&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>40.0x0.80 WATER objective</td>
			<td>Leica</td>
			<td>Leica HCX APO L U-V-I&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>25.0x0.95 WATER objective</td>
			<td>Leica</td>
			<td>HCX IRAPO L</td>
			<td></td>
		</tr>
	</tbody>
</table>

interfacing 3d engineered neuronal cultures to micro-electrode arrays: an innovative in vitro experimental model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

在此实验过程中，相应的作用是通过定义机械支架的三维神经网络的生长微珠起到图1A和B显示了微珠（排列的40±2微米的标称直径;认证平均直径42.3±1.1微米），在平面中。这种结构的特点是，微珠自发自组装限定紧凑六角形形状（图1B 和C）。如此生成的支架保证了持续增长的表面突起和胞体的代谢交换足够大的空隙空间。这种微珠的尺寸被证明是一个合理的...

Watch this Scientific Journal Video about Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model at JoVE.com

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

接口3D工程神经细胞微电极阵列：一种创新<em&gt;体外</em&gt;实验模型

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

interfacing-3d-engineered-neuronal-cultures-to-micro-electrode-arrays

Research

JoVE Journal

Neuroscience

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

9.9K 次观看.  University of Genova. In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

视频: Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

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

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

器官型培养的神经元雪崩多电极阵列记录

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

科研

神经科学

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

胚胎腹侧中脑器官型片文化：一个系统来研究多巴胺能神经元发展在体外</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

VisioTracker, an Innovative Automated Approach to Oculomotor Analysis

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, robust, and simple to manipulate. A suitable model has been found in the optokinetic response (OKR), a reflexive behavior present in all vertebrates due to its high selection value. The OKR involves slow stimulus-following movements of eyes alternated with rapid resetting saccades. The measurement of this behavior is easily carried out in zebrafish larvae, due to its early and stable onset (fully developed after 96 hours post fertilization (hpf)), and benefitting from the thorough knowledge about zebrafish genetics, for decades one of the favored model organisms in this field. Meanwhile the analysis of similar mechanisms in adult fish has gained importance, particularly for pharmacological and toxicological applications.
Here we describe VisioTracker, a fully automated, high-throughput system for quantitative analysis of visual performance. The system is based on research carried out in the group of Prof. Stephan Neuhauss and was re-designed by TSE Systems. It consists of an immobilizing device for small fish monitored by a high-quality video camera equipped with a high-resolution zoom lens. The fish container is surrounded by a drum screen, upon which computer-generated stimulus patterns can be projected. Eye movements are recorded and automatically analyzed by the VisioTracker software package in real time.
Data analysis enables immediate recognition of parameters such as slow and fast phase duration, movement cycle frequency, slow-phase gain, visual acuity, and contrast sensitivity.
Typical results allow for example the rapid identification of visual system mutants that show no apparent alteration in wild type morphology, or the determination of quantitative effects of pharmacological or toxic and mutagenic agents on visual system performance.

The VisioTracker is an automated system for the quantitative analysis of visual performance of larval and small adult fish based on the recording of eye movements. It features full control over visual stimulus properties and real-time analysis, enabling high-throughput research in fields such as visual system development and function, pharmacology, neural circuit studies and sensorimotor integration.

VisioTracker，创新的自动化到动眼神经分析方法

Investigations into the visual system development and function necessitate quantifiable behavioral models of visual performance that are easy to elicit, ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.

Primary disassociated embryonic hippocampal neuronal cultures are useful for investigating the signaling mechanisms involved in neuron death. Sexing the embryos before the isolation and dissociation of the hippocampus allows the preparation of separate male and female cultures, which enables the researcher to identify and investigate sex-specific cell signaling.

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

一个程序植入微丝的有组织阵列的单机录音清醒，举止动物

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Neural Activity Propagation in an Unfolded Hippocampal Preparation with a Penetrating Micro-electrode Array

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity in the hippocampus. The transverse hippocampal slice preparation is the most common tissue preparation to study hippocampus electrophysiology. A longitudinal hippocampal slice was also developed in order to investigate longitudinal connections in the hippocampus. The intact mouse hippocampus can also be maintained in vitro because its thickness allows adequate oxygen diffusion. However, these three preparations do not provide direct access to neural propagation since some of the tissue is either missing or folded. The unfolded intact hippocampus provides both transverse and longitudinal connections in a flat configuration for direct access to the tissue to analyze the full extent of signal propagation in the hippocampus in vitro. In order to effectively monitor the neural activity from the cell layer, a custom made penetrating micro-electrode array (PMEA) was fabricated and applied to the unfolded hippocampus. The PMEA with 64 electrodes of 200 &#181;m in height could record neural activity deep inside the mouse hippocampus. The unique combination of an unfolded hippocampal preparation and the PMEA provides a new in-vitro tool to study the speed and direction of propagation of neural activity in the two-dimensional CA1-CA3 regions of the hippocampus with a high signal to noise ratio.

We have developed an in vitro unfolded hippocampus which preserves CA1-CA3 array of neurons. Combined with the penetrating micro-electrode array, neural activity can be monitored in both the longitudinal and transverse orientations. This method provides advantages over hippocampal slice preparations as the propagation in the entire hippocampus can be recorded simultaneously.

神经活动的传播的展开海马准备具有穿透微电极阵列

This protocol describes a method for preparing a new in vitro flat hippocampus preparation combined with a micro-machined array to map neural activity 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 ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

体外楔形切片准备模拟在Vivvo神经元电路连接

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...