Name: design, surface treatment, cellular plating, and culturing of modular neuronal networks composed of functionally inter-connected circuits
Uploaded: 2015-04-15T13:00:00.000+00:00
Duration: 10 min 32 s
Description: Watch this Scientific Journal Video about Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits at JoVE.com

这项工作是由欧洲项目BRIANBOW（FP7-年轻探险家的支持，笔者想感谢雅格布Tessadori博士对稿件有用的意见，和Silvia Chiappalone为她制作的视频所使用的图形帮助。

该程序是根据美国国立卫生研究院的护理标准和使用实验动物的完成，并批准了特拉维夫大学动物护理和使用委员会（许可证号 - L-14-019）。 1.准备仪器的PDMS <ol><li>准备晶片（ 材料的表 ，或从一个微细加工实验室的晶片），手术刀和镊子-灭菌是不需要的。 </li><li>根据以下条件使聚-D-赖氨酸（PDL）溶液:4毫克/毫升的0.1M硼酸盐缓冲液，pH 8，并储存在-20℃。 </li><li>根据以下程序制备的PDMS模具: <ol><li>由基和以10比上述固化剂混合在一起制备聚二甲基硅氧烷（PDMS，聚硅氧烷弹性体）:1，然后搅拌2物质为大约5分钟。 </li><li>每次放置在真空室中两次15分钟，并确认气泡消失。 </li><li>什么时候与PDMS就绪，准备旋涂器:开放氮旋钮，以允许气体使用，放置在旋涂器在硅晶片（ 图1A），并使用真空，以防止其移动。 </li><li>倾PDMS超过晶片并激活离心器1分钟以1,000rpm（创建大约100微米的高度）。 </li><li>放置晶片的热板上在100℃下进行30分钟。 </li><li>当PDMS已硬化，勾勒出模板的边界与PDMS，使用巴斯德吸管。放置晶片的热板上在100℃下进行30分钟。 </li><li>当PDMS边界已经硬化，用手术刀根据边框刻蜡板，并从晶圆剥离硅模板（含单一模式的平方PDMS）。 </li></ol></li></ol> 2.培养皿和盖玻片准备<ol><li>准备23毫米方形玻璃盖玻片，并清除它们按以下顺序: <ol><li>用蒸馏水，70％的乙醇和丙酮洗涤。 </li><html><li>用异丙醇和快速干燥氮气清洗。 </li></ol></li><li>放在盖玻片图案的PDMS模具，并轻轻按压，以确认它们牢固地附着在玻璃表面。放置在真空室15分钟。 </li><li>掉在模板1毫升PDL的。插入盖玻片进入真空室两次，每次20分钟，并留下的PDL O / N晾干。 </li><li>准备培养皿中创建一个支持蜂窝网络: <ol><li>覆盖培养皿（3.5厘米）用1毫升的PDL的2小时中的RT的表面上。除去用吸管将PDL下降。 </li><li>用蒸馏水冲洗，并留下来干。放置一个非常小的滴硅脂上盖玻片的每个角。 </li></ol></li><li>将盖玻片上的培养皿（ 即 PDMS应朝上），然后按中央轻轻核实附件。 </li><li>轻轻地用小镊子从盖玻片删除PDMS。灭菌，暴露于紫外线照射7分钟。 </li></ol>乐"&gt; 3。多电极阵列（MEA）的制备<ol><li>清洁MEA在这个顺序（ 表材料 ）: <ol><li>水在自来水和超声的集中，酶洗涤剂洗3次。 </li><li>声处理的蒸馏水的3倍。 </li><li>用蒸馏水（在引擎盖，1000微升尖）3次并置于紫外光下30分钟洗涤。 </li></ol></li><li>支持网络准备: <ol><li>准备设计支持网络的模具。 <ol><li>倾PDMS成一个12孔板（22毫米直径）。 </li><li>当与PDMS硬化取出模具并使用手术刀，切孔在中间以创建一个环状。 </li><li>放置在MEA（ 图2A）的中心的设计支持网络的模具中，盖上PDL中的表面的其余部分进行2小时，在室温。 </li></ol></li><li>使用移液管除去PDL和洗涤用蒸馏水。 </li><li>取出模具，并留下干燥（ 图2A）。&lt;/ li&gt; </ol></li><li>对准蜡纸对于MEA以下列方式: <ol><li>模板放置在指定的微机器人。使用倒置显微镜，以精确对准图案化结构的电极，并降低模版，直到它被放置在MEA表面（ 图2B）。 注:带好清洗MEA联系提供了PDMS和MEA自身之间的竞争附着力。 </li><li>解除显微和如果需要的话，可以使用镊子应用上述的PDMS少量的压力，以防止它从MEA分离。 </li><li>轻轻按下模板来MEA表面，并用显微镜才能验证它是否连接牢固，并且完全一致。 （ 图2C-D）。 </li></ol></li><li>放置的MEA在真空室15分钟;把1毫升滴PDL对模板。 </li><li>插入到真空腔室2次，每次20分钟。离开PDL干O / N在孵化器。 </li><li>电镀前，从多边环境协定取出PDMS模具和洗用无菌瓦特亚特。在紫外光照射下放置7分钟。 </li></ol> 4.解剖和文化<ol><li>在赫尔佐格等2011 21介绍准备文化。 </li></ol> 5.电镀<ol><li>计算所需要的电镀的细胞数，使用血细胞计数器（根据电路的作为结果讨论大小最佳密度）。 <ol><li>确保计数室（血球）是干净的，在其上放置盖玻片（使用酒精清洗）。 </li><li>稀释10微升的细胞悬浮在190微升电镀介质（稀释1:20）的。 </li><li>负载10微升稀释的细胞上的计数室的边缘，并慢慢地吸取细胞进行允许该腔室，以填补本身。 </li><li>使用倒置显微镜，可视化的血球网格。确定细胞中通过直接计数室中数（健康的细胞应该是圆形）。算上中大型广场的无覆盖细胞UT斯达康那些过路的边缘。 </li><li>计算细胞的浓度: 细胞的总数目/ 1000微升=计数×稀释倍数×10 4（血细胞计数仪的面积）的总细胞。 注意:例如，如果稀释因子为20和计数细胞总数分别为100: 100×20×10 4 = 0.1×10 7细胞/ 1,000微升或1×106/100微升。为了制版0.75×10 6个细胞，取75微升出细胞。 </li></ol></li><li>取所需的每单MEA或培养皿电镀单元的数量，悬浮细胞，以防止聚集，并镀它们在中心处，在patternate区域的上方（如果需要的话，也可以以稀释的细胞在培养基中） 。对于在MEA，放置在中间100微升下降。对于盖玻片，放置在中间1000微升下降。 </li><li>孵育细胞铺板在37℃下40分钟。加镀介质向镀细胞，高达1每毫升MEA和每盖玻片2毫升。 </li><li>保持在37℃和，每4天，以稀释富含0.5青霉素 - 链霉素，2％的B-27和0.75％Glutamax的新鲜生长培养基。 </li><li>从6/7 DIV，看到岛屿之间的连接后，稀释培养基10微升/毫升FUDR（25毫克脱氧尿苷+ 62.5毫克尿苷在12.5毫升MEM（最低必需培养基Eagle））或任何其他抗有丝分裂剂，以防止神经胶质过度生长。 </li></ol>

<ol>
	<li>Buzsaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+syntax:+cell+assemblies,+synapsembles,+and+readers.">Neural syntax: cell assemblies, synapsembles, and readers.</a> Neuron. 68, 362-385 (2010).</li><li>Meunier, D., Lambiotte, R., Bullmore, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+and+hierarchically+modular+organization+of+brain+networks.">Modular and hierarchically modular organization of brain networks.</a> Frontiers in Neuroscience. 4, 200 (2010).</li><li>Levy, O., Ziv, N. E., Marom, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+neural+representation+capacity+by+modular+architecture+in+networks+of+cortical+neurons.">Enhancement of neural representation capacity by modular architecture in networks of cortical neurons.</a> European Journal of Neuroscience. 35, 1753-1760 (2012).</li><li>Berdondini, L., 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=A+microelectrode+array+(MEA)+integrated+with+clustering+structures+for+investigating+in+vitro+neurodynamics+in+confined+interconnected+sub-populations+of+neurons.">A microelectrode array (MEA) integrated with clustering structures for investigating in vitro neurodynamics in confined interconnected sub-populations of neurons.</a> Sensors and Actuators B-Chemical. 114, 530-541 (2006).</li><li>Bisio, M., Bosca, A., Pasquale, V., Berdondini, L., Chiappalone, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+bursting+activity+in+connected+neuronal+sub-populations.">Emergence of bursting activity in connected neuronal sub-populations.</a> PloS One. 9, e107400 (2014).</li><li>Shein Idelson, M., Ben-Jacob, E., Hanein, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+synchronous+oscillations+in+freely-organized+small+neuronal+circuits.">Innate synchronous oscillations in freely-organized small neuronal circuits.</a> PloS One. 5, e14443 (2010).</li><li>Shein-Idelson, M., Ben-Jacob, E., Hanein, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+neuronal+circuits:+a+new+platform+for+studying+the+role+of+modular+topology.">Engineered neuronal circuits: a new platform for studying the role of modular topology.</a> Frontiers in Neuroengineering. 4, 10 (2011).</li><li>Bonifazi, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+large-scale+experimental+and+theoretical+studies+for+the+realization+of+bi-directional+brain-prostheses.">In vitro large-scale experimental and theoretical studies for the realization of bi-directional brain-prostheses.</a> Front Neural Circuits. 7, 40 (2013).</li><li>Jungblut, M., Knoll, W., Thielemann, C., Pottek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triangular+neuronal+networks+on+microelectrode+arrays:+an+approach+to+improve+the+properties+of+low-density+networks+for+extracellular+recording.">Triangular neuronal networks on microelectrode arrays: an approach to improve the properties of low-density networks for extracellular recording.</a> Biomedical Microdevices. 11, 1269-1278 (2009).</li><li>Marconi, 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=Emergent+functional+properties+of+neuronal+networks+with+controlled+topology.">Emergent functional properties of neuronal networks with controlled topology.</a> PloS One. 7, e34648 (2012).</li><li>Taylor, A. M., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+and+compartmentalized+platforms+for+neurobiological+research.">Microfluidic and compartmentalized platforms for neurobiological research.</a> Critical Reviews in Biomedical Engineering. 39, 185-200 (2011).</li><li>Sorkin, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compact+self-wiring+in+cultured+neural+networks.">Compact self-wiring in cultured neural networks.</a> J Neural Eng. 3, 95-101 (2006).</li><li>Mata, A., Fleischman, A. J., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+polydimethylsiloxane+(PDMS)+properties+for+biomedical+micro/nanosystems.">Characterization of polydimethylsiloxane (PDMS) properties for biomedical micro/nanosystems.</a> Biomedical Microdevices. 7, 281-293 (2005).</li><li>Campo, A. G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SU-8:+a+photoresist+for+high-aspect-ratio+and+3D+submicron+lithography.">SU-8: a photoresist for high-aspect-ratio and 3D submicron lithography.</a> Journal of Micromechanics and MicroengineeringEmail alert RSS feed. 17, 81-95 (2007).</li><li>Liu, G. T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kan+Y+Fabrication+of+high-aspect-ratio+microstructures+using+SU8+photoresist.">Kan Y Fabrication of high-aspect-ratio microstructures using SU8 photoresist.</a> Microsystem Technologies. 11, 343-346 (2005).</li><li>Jackman, R. J., Duffy, D. C., Cherniavskaya, O., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+elastomeric+membranes+as+dry+resists+and+for+dry+lift-off.">Using elastomeric membranes as dry resists and for dry lift-off.</a> Langmuir. 15, 2973-2984 (1999).</li><li>Pine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+action+potentials+from+cultured+neurons+with+extracellular+microcircuit+electrodes.">Recording action potentials from cultured neurons with extracellular microcircuit electrodes.</a> Journal of Neuroscience Methods. 2, 19-31 (1980).</li><li>Gross, G. W., Rieske, E., Kreutzberg, G. W., Meyer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Fixed-Array+Multi-Microelectrode+System+Designed+for+Long-Term+Monitoring+of+Extracellular+Single+Unit+Neuronal-Activity+In+vitro.">New Fixed-Array Multi-Microelectrode System Designed for Long-Term Monitoring of Extracellular Single Unit Neuronal-Activity In vitro.</a> Neuroscience Letters. 6, 101-105 (1977).</li><li>Gross, G. W., Williams, A. N., Lucas, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+of+spontaneous+activity+with+photoetched+microelectrode+surfaces+from+mouse+spinal+neurons+in+culture.">Recording of spontaneous activity with photoetched microelectrode surfaces from mouse spinal neurons in culture.</a> Journal of Neuroscience Methods. 5, 13-22 (1982).</li><li>Thomas, C. A., Springer, P. A., Loeb, G. E., Berwald-Netter, Y., Okun, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+miniature+microelectrode+array+to+monitor+the+bioelectric+activity+of+cultured+cells.">A miniature microelectrode array to monitor the bioelectric activity of cultured cells.</a> Experimental Cell Research. 74, 61-66 (1972).</li><li>Herzog, N., Shein-Idelson, M., Hanein, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+validation+of+in+vitro+extra-cellular+neuronal+recordings.">Optical validation of in vitro extra-cellular neuronal recordings.</a> J Neural Eng. 8, 056008 (2011).</li><li>Maccione, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+algorithm+for+precise+identification+of+spikes+in+extracellularly+recorded+neuronal+signals.">A novel algorithm for precise identification of spikes in extracellularly recorded neuronal signals.</a> Journal of Neuroscience Methods. 177, 241-249 (2009).</li><li>Dworak, B. J., Wheeler, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+MEA+platform+with+PDMS+microtunnels+enables+the+detection+of+action+potential+propagation+from+isolated+axons+in+culture.">Novel MEA platform with PDMS microtunnels enables the detection of action potential propagation from isolated axons in culture.</a> Lab Chip. 9, 404-410 (2009).</li><li>Georger, J. H., 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=Coplanar+Patterns+of+Self-Assembled+Monolayers+for+Selective+Cell-Adhesion+and+Outgrowth.">Coplanar Patterns of Self-Assembled Monolayers for Selective Cell-Adhesion and Outgrowth.</a> Thin Solid Films. 210, 716-719 (1992).</li><li>Torimitsu, K., Kawana, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+Growth+of+Sensory+Nerve-Fibers+on+Metal-Oxide+Pattern+in+Culture.">Selective Growth of Sensory Nerve-Fibers on Metal-Oxide Pattern in Culture.</a> Developmental Brain Research. 51, 128-131 (1990).</li><li>Branch, D. W., Corey, J. M., Weyhenmeyer, J. A., Brewer, G. J., Wheeler, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microstamp+patterns+of+biomolecules+for+high-resolution+neuronal+networks.">Microstamp patterns of biomolecules for high-resolution neuronal networks.</a> Medical &amp; Biological Engineering &amp; Computing. 36, 135-141 (1998).</li><li>Petrelli, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-volume+drop+patterning+for+rapid+on-chip+neuronal+connect-ability+assays.">Nano-volume drop patterning for rapid on-chip neuronal connect-ability assays.</a> Lab on a Chip. 13, 4419-4429 (2013).</li><li>Boehler, M. D., Leondopulos, S. S., Wheeler, B. C., Brewer, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+networks+on+reliable+patterned+substrates.">Hippocampal networks on reliable patterned substrates.</a> Journal of Neuroscience Methods. 203, 344-353 (2012).</li></ol>

作者什么都没有透露。

的协议种植2D模块化神经网络的体外功能上相互连接组成的电路描述。该过程是基于图案的蜂窝的粘合剂层。图案形成用的PDMS模具再现期望网络体系结构的负特性来实现的。 PDMS模板定义，其中所述蜂窝粘合剂层沉积领域。一旦细胞被铺板，它们自发地装配到涂覆岛屿和自组织成活性间连接的电路。利用多边环境协定和钙成像记录功能模块化的网络录音提交。 所提出的协议已正式修改相比最初遵循的程序。首先，问题PDMS模板&#39;对齐必须得到解决。由于取向必须在第一次尝试实现（一旦模板已经碰了面，所以不建议搬迁的话），这个问题已经解决通过放弃手动程序，并选择加入一个新的方法，包括使用微操纵器上的MEA记录区精确的模具沉积。即必须解决的另一个问题在于防止PDL中滴从通过对准的模版下方（否则该图案设计不能得到）。这样做，一旦模版已经对齐，并把对PDL下降就可以了，所考虑的装置（MEA或盖玻片）之前，必须留在真空室中的时间的适当时间，从而提供了其上的一个良好的附着表面。同样已完成一次对PDL滴被放置在模版，以消除在设备和下降从而本身允许的PDL获取与所述表面接触之间形成的气泡。 i）该旋涂相应保证得到"开放复制品"或者，至少一个suffici:被所描述的协议的关键步骤由下式表示ently薄膜在SU8结构，可以使用镊子容易地除去; ii）所述培养表面（盖玻片或MEA）的治疗; 3）放置在其上的PDMS模具的; iv）该涂层相，因为有这样的情况其中，如果该PDMS掩模不能正常对在其上被定位在基板密封，它可能发生，该黏着蛋白通过下部的掩模从而影响该构图。所以，盖单或的MEA的清洗过程是最重要的，以及与PDMS模版的对准。这两个步骤适当演出确保最佳构图的结果。 这种技术的局限性也有做与两者电和钙成像采集。在第一种情况下，非常低的空间分辨率（4 - 8电极被包括在每个模块中）的存在，由于单回路的两个尺寸和电极间distanc即在第二种情况下，风险在于未找到最佳蜂窝密度，它允许获得一个二维细胞层，从而防止单细胞分辨率采集。 这一策略，即要求涂装，总装和消毒程序，创建一个自定义的微准系统的仔细优化，给了我们一些主要的优点相对于此前报道的作品（ 如，我们的细胞的长期生存， 即，最多至8周体外 ）。具体地讲，它为我们提供了更稳定可靠的长期禁闭24相对于基于化学构图23-28策略。 所描述的过程依赖于PDL涂覆模块，这保证了自组织网络的进入由轴突连接的神经元组件和树突束镀皮质细胞。以允许模块化净的宽视场钙成像可与单细胞分辨率，至关重要的是，神经系统组织成一个二维层。因此，这是至关重要的，以优化镀细胞的密度既避免高密度，细胞群集或在哪些减少模块间连接概率的另一种极端的低密度模块。 虽然在培养神经细胞-胶质网络失去其生物体内结构组织，它们自发地自组织成活性功能电路提供进行良好控制的实验条件下，研究的可能性。在这个协议中描述的功能上相互连接的电路构成的二维模块化神经网络进行调查的通信中的神经元组件的动力学和结构上定义的神经元网络的能力，以产生基序的大型剧目提供了有力的工具。

实验和理论的证据支持，大脑运作通过的单元组件1-5协调活化，这可以被视为动态功能单元瞬时彼此，整形和下层不同大脑状态相互作用的可能性。官能模块化还依赖于与大脑电路6,7的结构的模块化组织关联。怎样的功能和大脑回路的结构互相塑造彼此仍是主要的开放性问题中的神经之一。以提供对这个问题更深入的了解，来识别最佳实验框架，其中能够解决，至少部分地，这些问题是重要的。由于控制操作的神经元网络的时空动力 ​​学在体内实验是具有挑战性的， 在体外的神经元网络模型的开发是显著感兴趣的，因为它们容易ACCessibility，监控，处理和建模8,9。在最近几年， 在由先进衬底图案化的方法支持体外技术已经允许诱导神经元网络开发一系列预定义的模块化结构体3，并与所施加的拓扑结构10学习网络的功能特性。特别是，方法进行了最近使用过的征收物理约束4,11组织网络。的确，以研究在神经元网络的结构和功能之间的联系，并提供相互作用的神经元组件的简化，但可能的表述， 在体外系统应提供相互连接的神经元亚群。广泛的研究二维均匀的神经细胞也没有对电路的自组织紧急连线任何空间的限制。因此，一个可能的方法来塑造人为地相互连接的电池组件被定位在不同的吐口水的神经元群体ially不同的领域。这些区域之间的距离不阻止除装配连接。这种方法，同时保证有相当的控制网络的复杂性，已经显示出，以提供同步模型6,7,12的更丰富的剧目。 为了方便的模块化神经元组件的可重复培养，协议组装的自组织网络到由轴突和树突连接的神经元集群的呈现和描述。聚合物结构对神经元培养物的物理约束已创建从polydimtheylsiloxane（PDMS）。 PDMS是一种弹性体广泛用于由于其生物相容性，透明性和透气性13生物医学应用。将PDMS制备并排除在微加工的SU8 2075 14,15的结构，通过旋涂液态PDMS上如杰克曼等人以前描述了"主"。16吨他实现了图形化的神经网络是由不同大小的相互连接的模块和他们两个盖玻片和微电极阵列（多边环境协定）17-20成功获得。在模块之间连接的密度可以改变它的网络同步的功能，从一个完全同步的网络中，典型的均一培养，以同步的瞬时状态模块之间。

<table><tbody><tr><td>PDMS, Sylgard 184</td><td>Dow Corning</td><td></td><td></td></tr><tr><td>Nalgene vacuum chamber</td><td>Thermo</td><td>5305-0609</td><td></td></tr><tr><td>Poly-D-lysine PDL</td><td>Sigma</td><td>P7886</td><td></td></tr><tr><td>Silicone grease - SILICAID 1010</td><td>aidchim Ltd</td><td>H3375</td><td></td></tr><tr><td>Spin coater</td><td>Laurell - Technologies Corporation</td><td>WS-650-23</td><td></td></tr><tr><td>12-well culture plate</td><td>Sigma</td><td>CLS3336</td><td></td></tr><tr><td>5-Fluoro-2&amp;rsquo;-deoxyuridine</td><td>Sigma</td><td>F0503</td><td></td></tr><tr><td>Uridine</td><td>Sigma</td><td>U3003</td><td></td></tr><tr><td>MEA1060-Inv-BC</td><td>Multi Channel Systems</td><td></td><td></td></tr><tr><td>TC02</td><td>Multi Channel Systems</td><td></td><td></td></tr><tr><td>Pen Strep</td><td>Biological Industries Beit Haemek</td><td>03-033-1c</td><td></td></tr><tr><td>B-27</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>glutaMAX</td><td>Gibco</td><td>35050-038</td><td></td></tr><tr><td>MEM Minimum Essential Medium-Eagle</td><td>Biological Industries Beit Haemek</td><td>01-025-1B</td><td></td></tr><tr><td>Micro Electrode Arrays 4Q</td><td>Multi Channel Systems</td><td>60-4QMEA1000iR-Ti-pr</td><td>cleaning manual: &lt;a href=&quot;http://www.multichannelsystems.com&quot; target=&quot;_blank&quot;&gt;http://www.multichannelsystems.com&lt;/a&gt;</td></tr><tr><td>Silicon wafer</td><td>microchem</td><td>SU8-2075</td><td>Preparation protocol: &lt;a href=&quot;http://www.microchem.com&quot; target=&quot;_blank&quot;&gt;http://www.microchem.com&lt;/a&gt;</td></tr></tbody></table>

design, surface treatment, cellular plating, and culturing of modular neuronal networks composed of functionally inter-connected circuits

大脑工作通过协调激活和神经元组件的动态通信。一个主要的开放问题是如何动态图案​​的巨大的剧目，这背后最多样化的大脑功能，可以浮现出的大脑回路的固定拓扑和模块化的组织。相比对神经元回路的体内研究，其呈现固有的实验的困难， 在体外制剂提供了更大的可能性，以操纵和探针实验神经系统的结构，动力学和化学性质。这项工作描述了在体外实验方法，它允许增长了空间层次分明，功能上相互联系的神经元组成的组件模块化网络。该协议允许控制神经网络的二维（2D）结构在不同级别的复杂性拓扑。 所期望的网络图案可定期盖玻片和基材嵌入式微电极阵列来实现两者。微机械结构被压印在硅晶片上，并用于创建生物相容性聚合物模具，其包含所述期望的网络架构的负特征。刻花模板与一个分子层用于促进细胞粘附的表面涂层过程期间放置在培养底物。除去模板后，神经元被镀和它们自发地重定向到涂覆区域。通过减小隔室间的距离，因此能够获得任何分离的或互相连接的神经元回路。促进细胞存活，细胞共培养，其位于培养皿的周围的支撑神经元网络。分别通过使用基板嵌入式微电极阵列和钙成像获得模块化网络的活动的电和光学记录被呈现。虽然每个模块显示SPONTaneous全球同步，对模块间同步的发生是由电路之间连接的密度调节。

甲SU8-2075模具的硅晶片具有约100μm的厚度的功能上是用来塑造的PDMS。的图案是由多个维度的平方，具有200至700微米（ 图1B）的侧的长度和距离变化。被选为广场的大小以适合的观点10X的字段（岛屿与一个边长&lt;800微米）和20X目标（对于岛屿边长&lt;400微米）。三个参数，电路之间即细胞铺板密度，距离，电路&#39;的大小是决定用于得到单层或聚集电路以及强加和形状的连接。然而，这些参数不是自独立，给定的两个电路之间的固定距离，它们自发地建立连接的增加与电路"大小的概率。在这项工作中所讨论的，对于〜300的小神经元模块的例子×300微米，连接成立时的距离不超过300〜较大 - 400微米，而对于大型神经模块〜700×700微米，连接建立时的距离为小于〜700微米。在一般情况下，通过增加的平方之间的距离是可以改变以建立模块间自发产生间的连接​​的概率，从高度连接的模块传递到孤立的。 的〜600×600微米的体外图示，以4天的神经元模块（DIV; 图3A）。后在体外的神经元几天大多分布在涂层区域内，而这是不可能观察发达神经元突起和连接。与此相反，在14格（ 图3B-C）的神经元内建立和模块间连接。虽然〜600×600微米的较大的神经元回路可自组织成单分子膜（ 图3A-C）小了的，电路R角尺寸（ 例如，〜300×300微米， 如图3D）趋于聚集。因此，为了获得最佳校准二维电路，将细胞铺板与来自附着在35毫米的培养皿中0.25×10 6〜1×10 6个细胞/ 23毫米遮滑移不同密度不同。对于较大的〜700×700微米的电路我们发现，附着在35毫米的培养皿中0.75×10 6细胞/ 23毫米遮滑移是诱导的单层电路不防止其自发间连接形成的最优密度（ 图3C ）。在〜300×300微米的较小的电路通过电镀附着在35毫米的培养皿0.5×10 6个细胞/23毫米盖玻片得到单层电路的相同的结果。在一般情况下，考虑到细胞铺板密度时，它强调了重要的是，如前面所讨论7，神经元和神经胶质细胞具有一种天生的倾向聚集，因此在一定程度CL的ustering是在培养一段时间几乎是不可避免的。然而，我们观察到，增加发往支持网络的区域将提高电路的生存和它们的单层组织的概率，可能是由于营养物在细胞外空间中的较大的浓度。在任何情况下，反复试验的方法是必需的，以确定最佳的细胞铺板密度创建用于钙成像单层电路。每4天将培养基稀释用新鲜生长培养基中。从6/7 DIV，看到岛之间的连接后，加入10μl/ ml的FUDR已被添加，以防止神经胶质增生。 图4示出了使用钙成像（如在Bonifazi 等人 2013 8所述进行）模块化网络的动态记录。使用4X放大倍数的物镜，这使得监测整个网络的活动被收购钙FL uorescence图像（ 造型玩具Ë4A）。如在Bonifazi 等人 2013 8所述进行的钙成像的分析。在钙事件图4B光栅情节起病显示自发活动呈现（颜色对应于标示图4A模块）。以30Hz进行钙成像和图像尺寸为1000×1000个像素。 绿色和粉红色的模块高度同步所示的光栅图 （ 图4B）中的协议与它们连接厚束，有可能对应于大量连接的（ 图4A）。蓝色和红色的模块是弱连接到绿色和粉红色的模块，因此它们偶尔与它们（ 图4B）同步。录制的视频可在网上（电影M1）。 在21 DIV生长在MEA模块化网络的图像示于图5A。我n阶以获得二维电路最好校准，我们镀皮质细胞与来自250×10 3个不同的密度变化至500×10 3个细胞/每MEA。 250×10 3个细胞/每MEA（30毫米环）的单层的电路的形成方面得到更好的结果，而不是防止其自发相互连接。一旦图案化神经网络已经达到上的MEA基板（ 图5A），电生理学活性已经使用用于采集和记录电生理信号的商用系统进行监控。使用精确定时棘波检测（PTSD）的算法22检测文化的自发活动。 图5B示出了对应于5分钟的自发活动的（唯一活性电极是可见的）栅格情节，着色根据簇号编码。通过观察这些图，它有可能使质量评估的同时，整个网络的单个簇和活性。尤其是，它有可能观察到每个集群内记录的活动的强烈的同步。具有代表性的记录超过MEA视频从图案的文化可以在线（电影M2）。 <img alt="图1" src="/files/ftp_upload/52572/52572fig1highres.jpg" width="700" /> 图1.硅晶片。 （A）中的硅晶片（〜4英寸直径），用于模制的PDMS模具。不同的SU8结构代表不同的模块化的网络。（B）中的硅晶片为模块化网络结构上实现代表特征的设计。每个绿点限定的SU8立柱结构，因此，该区域为一个单一的模块。虚线黑色矩形内的设计，对应于三种不同的模板，可以在为23mm侧len个标准的广场文化盖玻片使用GTH。在每个模板中，星号标记的宏区域的支持网络。根据光点之间的距离，所述有限大小的电路或模块可能能够建立它们之间自发的神经元的连接。的特征的设计被选择以创建不同的模块的大小和模块间的距离，以实现分离的或互相连接的神经元回路可能在不同的目标放大率适应的视场（4X，10X和20X的视每个放大率字段是由红色方块表示）。功能出了虚线黑色矩形的设计，以适应同时使用MEA和钙成像。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52572/52572fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52572/52572fig2highres.jpg" width="700" /> 图2。PDMS结构沉积在MEA。 （A）中的PDMS模具具有环形形状（标有红色箭头）安装在MEA。一个PDMS模具的模具被用于涂覆发往支持神经网络和位于该培养区域的周围的区域（这是由环限定安装在MEA和由绿色箭头标记）。（B）的比对在MEA用微型操纵器（℃）的PDMS模具沉积在上对准之后MEA。在使用10倍的放大倍率的MEA一个PDMS模版的（D）中的图像（电极间距离为500μm）。所以能够注意到在模具上的孔中的电极的对应关系。粘合层有利于细胞粘附会沉积仅在打开的地方。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52572/52572fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> GE ="总是"&gt; <img alt="图3" src="/files/ftp_upload/52572/52572fig3highres.jpg" width="700" /> 图在不同的DIV 3.模块化神经网络。的〜600×600微米（A）后，4 DIV和（B）后14 DIV。（C）皮质模块亮场图像后，14 DIV注意电路间的自发互连。细胞以最佳细胞密度为大电路组织成单层， 即，750×10 3个细胞附着在35毫米的培养皿/23毫米盖玻片。（D）的使用半角和相同的细胞浓度的PDMS特征，组织成集群结构的神经回路。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52572/52572fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> highres.jpg"宽度="700"/&gt; 图4.钙模块化网络的成像。装载了钙指示剂OGB（A）皮质细胞（18 DIV）。不同的颜色标记不同的模块。视场为2×2毫米的自发活动的（B）光栅情节。不同的颜色对应不同的模块内的细胞显示面板A. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52572/52572fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52572/52572fig5highres.jpg" width="700" /> 图5. MEA模块化网络的录制。 （一）生长在4Q MEA 3个不同的电路组成的模块化网络（皮层神经元，21 DIV）。神经元回路是遥远〜600微米彼此互连。（B）的光栅积表示5分钟的自发ELECTR的ophysiological活动记录。 C1，C2和C3相对应，分别左上角，右下角和左下角集群的活动，以不同的颜色突出显示。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52572/52572fig5highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits at JoVE.com

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

这手稿描述了一个协议， 在体外模块化网络包括空间上的限制，在功能上相互连接的神经回路的增长。的聚合物掩模被用来图案的蛋白质层，以促进通过培养基底细胞粘附。镀神经元生长在涂层区域自发建立连接并显示出电活性。

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 ...

design-surface-treatment-cellular-plating-culturing-modular-neuronal

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.4K 次观看.  Tel-Aviv University. 这手稿描述了一个协议， 在体外模块化网络包括空间上的限制，在功能上相互连接的神经回路的增长。的聚合物掩模被用来图案的蛋白质层，以促进通过培养基底细胞粘附。镀神经元生长在涂层区域自发建立连接并显示出电活性。 

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

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

3D Cocultures 星形胶质细胞与人多潜能干细胞神经元的突触电路建模

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

科研

发育生物学

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

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays (MEAs)

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop working in various diseases. Studies have included neuropathological observation, fluorescent microscopy with genetic labeling, and intracellular recording in both dissociated neurons and slice preparations. This protocol discusses another technology, which involves growing dissociated neuronal cultures on micro-electrode arrays (also called multi-electrode arrays, MEAs).
There are multiple advantages to using this system over other technologies. Dissociated neuronal cultures on MEAs provide a simplified model in which network activity can be manipulated with electrical stimulation sequences through the array's multiple electrodes. Because the network is small, the impact of stimulation is limited to observable areas, which is not the case in intact preparations. The cells grow in a monolayer making changes in morphology easy to monitor with various imaging techniques. Finally, cultures on MEAs can survive for over a year in vitro which removes any clear time limitations inherent with other culturing techniques.1
Our lab and others around the globe are utilizing this technology to ask important questions about neuronal networks. The purpose of this protocol is to provide the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs

This protocol provides the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.

如何以文化，记录和刺激神经网络的微电极阵列（多边环境协定）

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop ...

生物学

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.

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 ...

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 ...

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

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 accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

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 ...

Use of Pre-Assembled Plastic Microfluidic Chips for Compartmentalizing Primary Murine Neurons

Microfabricated methods to compartmentalize neurons have become essential tools for many neuroscientists. This protocol describes the use of a commercially available pre-assembled plastic chip for compartmentalizing cultured primary rat hippocampal neurons. These plastic chips, contained within the footprint of a standard microscope slide, are compatible with high-resolution, live, and fluorescence imaging. This protocol demonstrates how to retrograde label neurons via isolated axons using a modified rabies virus encoding a fluorescent protein, create isolated microenvironments within one compartment, and perform axotomy and immunocytochemistry on-chip. Neurons are cultured for &gt;3 weeks within the plastic chips, illustrating the compatibility of these chips for long-term neuronal cultures.

This protocol describes the use of plastic chips to culture and compartmentalize primary murine neurons. These chips are preassembled, user-friendly, and compatible with high-resolution, live, and fluorescence imaging. This protocol describes how to plate rat hippocampal neurons within these chips and perform fluidic isolation, axotomy and immunostaining.

预组装塑料微流体芯片在原生小鼠神经元分割中的应用

Microfabricated methods to compartmentalize neurons have become essential tools for many neuroscientists. This protocol describes the use of a ...

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a pre-assembled multi-compartment chip made in a cyclic olefin copolymer (COC) to compartmentalize neurons differentiated from human stem cells. The footprint of these COC chips are the same as a standard microscope slide and are equally compatible with high resolution microscopy. Neurons are differentiated from human neural stem cells (NSCs) into glutamatergic neurons within the chip and maintained for 5 weeks, allowing sufficient time for these neurons to develop synapses and dendritic spines. Further, we demonstrate multiple common experimental procedures using these multi-compartment chips, including viral labeling, establishing microenvironments, axotomy, and immunocytochemistry.

This protocol demonstrates the use of compartmentalized microfluidic chips, injection molded in a cyclic olefin copolymer to cultured neurons differentiated from human stem cells. These chips are preassembled and easier-to-use than traditional compartmentalized poly(dimethylsiloxane) devices. Multiple common experimental paradigms are described here, including viral labeling, fluidic isolation, axotomy, and immunostaining.

预组装塑料微流体芯片中人类干细胞衍生神经元的分段化

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a ...

HD-MEA 上的多模态大规模神经记录和分析 

Recording and Analyzing Multimodal Large-Scale Neuronal Ensemble Dynamics on CMOS-Integrated High-Density Microelectrode Array

Large-scale neuronal networks and their complex distributed microcircuits are essential to generate perception, cognition, and behavior that emerge from patterns of spatiotemporal neuronal activity. These dynamic patterns emerging from functional groups of interconnected neuronal ensembles facilitate precise computations for processing and coding multiscale neural information, thereby driving higher brain functions. To probe the computational principles of neural dynamics underlying this complexity and investigate the multiscale impact of biological processes in health and disease, large-scale simultaneous recordings have become instrumental. Here, a high-density microelectrode array (HD-MEA) is employed to study two modalities of neural dynamics - hippocampal and olfactory bulb circuits from ex-vivo mouse brain slices and neuronal networks from in-vitro cell cultures of human induced pluripotent stem cells (iPSCs). The HD-MEA platform, with 4096 microelectrodes, enables non-invasive, multi-site, label-free recordings of extracellular firing patterns from thousands of neuronal ensembles simultaneously at high spatiotemporal resolution. This approach allows the characterization of several electrophysiological network-wide features, including single/-multi-unit spiking activity patterns and local field potential oscillations. To scrutinize these multidimensional neural data, we have developed several computational tools incorporating machine learning algorithms, automatic event detection and classification, graph theory, and other advanced analyses. By supplementing these computational pipelines with this platform, we provide a methodology for studying the large, multiscale, and multimodal dynamics from cell assemblies to networks. This can potentially advance our understanding of complex brain functions and cognitive processes in health and disease. Commitment to open science and insights into large-scale computational neural dynamics could enhance brain-inspired modeling, neuromorphic computing, and neural learning algorithms. Furthermore, understanding the underlying mechanisms of impaired large-scale neural computations and their interconnected microcircuit dynamics could lead to the identification of specific biomarkers, paving the way for more accurate diagnostic tools and targeted therapies for neurological disorders.

作者聚焦：通过 HD-MEA 技术推进大规模神经动力学

Here, we employ HD-MEA to delve into computational dynamics of large-scale neuronal ensembles, particularly in hippocampal, olfactory bulb circuits, and human neuronal networks. Capturing spatiotemporal activity, combined with computational tools, provides insights into neuronal ensemble complexity. The method enhances understanding of brain functions, potentially identifying biomarkers and treatments for neurological disorders.

CMOS集成高密度微电极阵列上多模态大规模神经元集成动力学的记录与分析

Large-scale neuronal networks and their complex distributed microcircuits are essential to generate perception, cognition, and behavior that emerge from ...

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

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此视频中的章节

3D Cocultures 星形胶质细胞与人多潜能干细胞神经元的突触电路建模

神经元共培养与单细胞精度的制备

如何以文化，记录和刺激神经网络的微电极阵列（多边环境协定）

接口3D工程神经细胞微电极阵列:一种创新

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

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

预组装塑料微流体芯片在原生小鼠神经元分割中的应用

预组装塑料微流体芯片中人类干细胞衍生神经元的分段化

CMOS集成高密度微电极阵列上多模态大规模神经元集成动力学的记录与分析

CMOS集成高密度微电极阵列上多模态大规模神经元集成动力学的记录与分析