作者感谢乌尔里希Marggraf（ISAS）的SU-8制作和玛丽亚·贝克尔（ISAS）的扫描电镜成像。这项研究是由财政德意志研究联合会（DFG WE3737/3-1），一个Bundesministerium献给教化UND Forschung补助（BMBF 0101-31P6541）及的ministerium献给创新，Wissenschaft UND Forschung des Landes酒店北莱茵 - 威斯特法伦支持。海克Hardelauf感谢国际莱布尼茨研究生院“系统生物学实验室在一个芯片”的金融支持。

该协议适用于神经科学家，并总结在图2。因此，它是推荐使用的PDMS复制的SU-8的主人是微细加工外包给商业或公共机构设施。这两个图层蒙版的设计是可自由查看的是对化学网站12皇家学会补充资料（邮政编码）。重要的是，第一SU-8层应该被制造为2.5-3.0微米的深度，并且第二至25-30微米的深度。这些都是必要的有效的神经元排列关键维度。在3微米的高度限制也是必要的，以防止神经元被输送或迁移的两个隔室12之间。 在PDMS 1。微流控设备复制<ol><li>与固化剂（184）充分混合的PDMS预聚物以10:1的比率，并在真空干燥器中通常20分钟脱气混合物。另外，把混合TURE在50毫升Falcon管中，并使用低克 离心脱气。 </li><li>将2层的微结构的SU-8的晶片在80℃下加热板和对准聚合物帧到晶片上的每个设备。倾PDMS入帧≥5毫米的深度，并允许&gt; 1小时以确保完全热固化。 </li><li>一旦固化，从电炉取出晶片，并使其冷却在一个平面上。 </li><li>与刚性手术刀轻轻撬开框架和PDMS从硅片佩戴防护眼镜和工作从一个角（离SU-8微结构）。 </li><li>从框架中取出硅橡胶模具，并使用剪刀来修剪多余的PDMS的设备。这个所谓的闪烁通常时产生的PDMS薄膜的聚合物框架和晶片表面之间传播。 </li><li>其余的PDMS可以应用到整个晶片和固化存储期间保护晶片。之前，后续的设备成型取出这一点，并在这样做去除任意p从表面上表达的污染物。 </li></ol> 2，设备组装和互连<ol><li>使用3毫米直径的活检穿孔准备6接口端口。在一个漆黑的表面工作，在良好的光线条件和朝上，以帮助端口对准与微流体通道的微结构化特征。 </li><li>检查装置，用于微粒。使用可逆胶带移除。 </li><li>微流体回路是使用安装在玻璃盖玻片上的薄的PDMS层包封的：这些是​​由浇注PDMS的固化剂混合物（10:1，601）的一小体积（约0.5毫升）上的柔性基底制备细菌学级培养皿。轻轻按压盖玻片上的PDMS层，放在烤盘和热固化几分钟。快速冷却后，用手术刀切出和奖金盖玻片-PDMS双层从培养皿中。如有必要，用剪刀修剪。 </li><li>等离子债券的PDMS设备和支撑层，以产生良好的密封：最佳条件是特定的仪器。例如，等离子炉中在70瓦和40千赫在0.2毫巴的氧气氛中进行40秒的操作产生优秀的PDMS-PDMS键。一旦等离子体处理，可以同时按下两个部分组合在一起，留下了一分钟，充分结合。 </li><li>使用硅胶管（1.6 mm内径;3.2毫米OD）与泵和注射器接口。 </li></ol> 3，生物材料和图案化微流控操作 原位生物材料图案化，神经元培养和流体处理的协议为示于图2。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51389/51389fig2highres.jpg" width="500" /> 图2。画报微流体协议。 A）PL 和PLL-G-PEG图案 ;此外，细胞的粘附层（ 例如 ，PL，绿色），与蒸发产生对准水面膜二）暴露PL的大气等离子体处理;两个天线引脚使用，用图示用粉红色的明星。C）的PBS（蓝色）由抽吸冲洗是用来避免污染等离子体处理过的区域用PL D）交货PLL-G-聚乙二醇（红色的等离子导入天线）由愿望外套等离子体处理过的区域E） 小区负荷 ;神经元的同步微排列到两个侧翼舱吸，采用了静压驱动流G） 流体隔离治疗 女性）媒体（粉红色）灌注;测试剂（黑色）的外周治疗误吸和H）由吸入底部的中央进气送到中央处理从底部旁侧入口交付。图与传奇再现英国皇家化学学会的许可（RSC）12。 <a href="https://www.jove.com/files/ftp_upload/51389/51389fig2highres.jpg" target="_blank">点击这里查看大图。</a> 紧随等离子体结合的PDMS微流体通道具有高度的亲水性（接触角≤5°）。这样的表面是适合的细胞系，例如分化的人SH-SY5Y神经元样细胞的粘附和培养。为原代神经元和神经元前体细胞系，如隆德人类脑细胞系（LUHMES）多胺涂层，无论是聚赖氨酸（PL）或聚鸟氨酸（PO）的培养物的离体培养，需要作为静电锚。除了粘附蛋白如层粘连蛋白或纤连蛋白通常需要作为整联接口涂层。然而，PL和PO涂料容易结合神经，阻碍了微流体输送到数组的网站，也不必要施加剪切应力。为了解决这个问题，在芯片上的生物材料图案是需要材料的粘附在陷阱站点和生长微本地化。这也促进了室间连接的发展，降低当地的纠缠。该协议还包括添加的PEG材料以相邻区域，以确保蛋白质和神经元长的培养过程中被限制到所需的位置。下面的协议是记录在图3： <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51389/51389fig3highres.jpg" width="500" /> 图3。水等离子体镂花掩蔽的生物材料的图案。 A）插图水掩模工艺与PDMS micropillars钉扎水半月板到位等离子镂花的。在垂直平面内的弯液面的曲率已被忽略不计。 乙）水掩模由弯月p可视化用红色染料和定位一局。 三）图案PL-FITC涂层等离子制版。经过D成像）增加蛋白质的排斥和细胞驱蚊PLL-G-PEG-TRITC到等离子图案PL-FITC涂层。这是用于SH-SY5Y细胞的登记和也用于图案化的附加层粘连蛋白为LUHMES细胞。图与传奇再现英国皇家化学学会（RSC）的许可12。 <a href="https://www.jove.com/files/ftp_upload/51389/51389fig3highres.jpg" target="_blank">点击这里查看大图。</a> <ol><li>水屏蔽原位生物材料图案等离子喷码机<ol><li> 0.5μL体积的PL或PO（100微克/毫升的1X PBS）的进入底部中心港口后不久等离子体结合吸管。在几秒钟内物质在整个微流体回路通过毛细管作用填充。离开了几分钟，充分外套亲水性的PDMS表面的多聚腺苷酸地雷。 </li><li>通过抽吸驱动的洗涤用1×PBS去除未结合的聚胺分子（参见图2C），持续1分钟。 </li><li>从端口卸下所有的PBS和照明用显微镜加热装置。这从快速补水面膜形成的端口增加了蒸发。水掩模（ 如图3A所示，并记录在图3B）被内〜5分钟建立。 </li><li>通过显微镜检查设备，以确保水的屏蔽是完整的。请注意储器结构阵列位点的上游包括在掩模设计来延长水掩模（〜15分钟）的稳定性。 </li><li>插入不锈钢针插入口（ 图2B）。这些被用来偶联通过常压手持式电晕放电或特斯拉发生器产生的等离子体（ 例如 ，运行在2兆赫，30千伏信号）19,20到空微流体通道。点T的前端他等离子体源接近1针和血浆的前端处理微通道为≤1秒。使用低光照条件下，观察血浆图案。 </li><li>用洗涤步骤去除水中的面具。从上面3个端口使用并行愿望。使用硅胶管由4路转接器抽吸泵连接4相同长度，实现并行愿望。开始抽吸和分配的PBS进入底部3个端口通过微流体电路绘制的PBS。以这种方式PDMS-聚胺材料的对比度产生（参见图3C）。从微流体电路撤离的PBS，离开几个小时，以恢复原生，疏水状态。这使得该PDMS完全非允许细胞粘附。 </li><li>应该附加的粘附材料，如层粘连蛋白或纤连蛋白，需要除去水掩模之后立即需要执行以下的步骤和要做到：移液器的接枝共聚物聚-L的10微升体积-赖氨酸-聚（乙二醇）（PLL-G-PEG，100微克/毫升，溶于PBS中）到两个底部侧翼通道，接着从2分钟的上侧翼通道抽吸。 </li><li>在不中断的流动，通过用PBS缓冲液交换除去过量的PLL-G-PEG。这个选择性地涂覆在非聚胺涂覆的区域用PEG部分作用，以防止蛋白质吸附和细胞粘附17,21。 </li><li>粘附蛋白如层粘连蛋白或纤连蛋白可以被共同定位与聚胺涂层：这些ECM蛋白（通常为10微克/毫升）的移液管加入10μl体积为所有三种底部端口。吸出物从顶部的三个端口，直到该设备填充有蛋白溶液孵育1小时，以产生良好的品质的涂层。 </li><li>通过从底部3口吸的PBS中取出多余的蛋白质。 </li></ol></li><li>微流控单神经元组阵<ol><li>总理与批设备吃了媒体。加入20微升体积的底部的3个端口，并通过从上三个端口并行抽吸迅速填满微流电路与媒体。通过等长的硅胶管采用4路油管接头耦合的3上端口的愿望泵。 </li><li>加入20微升的分类细胞悬液（1×10 6个细胞/ ml）以每一个侧翼吸入口（0，在图1中）。 </li><li>装备一个愿望泵流量调节器或使用注射器从上3个端口温柔的愿望。完整的神经元排列通常需要1分钟。 </li><li>收获残留在用移液管，用于排放在随后的微流体装置的4口侧翼过量的神经元。 </li><li>拆下油管。填写所有端口，媒体和设备放置在孵化器。在几个小时的细胞变得完全附着在生物材料上的图案。 </li></ol></li><li>神经元培养，选择性射流治疗和免疫nostaining <ol><li>通过定期灌注用流体静力进（2毫米柱的高度差是适当慢）交换媒体。 </li><li>可替换地，浸没在介质中的微流体装置中，定期地交换介质（2-3天），与标准的细胞培养物。 </li><li>选择性的流体处理，以任一神经元培养室或中央神经突向外生长隔室被实现如下：取20微升的测试物质的进入所关心的信道的底部端口，并从该端口的正上方抽吸。为冗长的治疗减少使用流量调节器的流量，并根据需要补充试验物质。 </li><li>免疫组化协议各有不同，每个分子靶点和相关试剂。为了反击的剪切诱导损伤缓慢流速使用的神经细胞的潜在风险。这会导致增加的应用时代，充分灌注系统，延长正常的协议倍。 HY使用的端口具有不同柱高度（ 例如 ，2毫米）drostatic灌注用于传递试剂。更快速试剂的添加和删除用泵可以用来减少实验的时间尺度。连接一个愿望泵上部3口从底部3个端口吸取试剂。 </li></ol></li></ol>

<ol>
	<li>Campenot, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+control+of+neurite+development+by+nerve+growth+factor.">Local control of neurite development by nerve growth factor.</a> Proc Natl Acad Sci USA. 74 (10), 4516-4519 (1997).</li><li>Campenot, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+sympathetic+neurons+in+compartmentalized+cultures.+I.+Local+control+of+neurite+growth+by+nerve+growth+factor.">Development of sympathetic neurons in compartmentalized cultures. I. Local control of neurite growth by nerve growth factor.</a> Dev Biol. 93 (1), 1-12 (1982).</li><li>Taylor, A. 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=Microfluidic+multicompartment+device+for+neuroscience+research.">Microfluidic multicompartment device for neuroscience research.</a> Langmuir. 19, 1551-1556 (2003).</li><li>Taylor, A. 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=A+microfluidic+culture+platform+for+CNS+axonal+injury,+regeneration+and+transport.">A microfluidic culture platform for CNS axonal injury, regeneration and transport.</a> Nat Methods. 2 (8), 559-565 (2005).</li><li>Kilinc, 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=Wallerian-like+degeneration+of+central+neurons+after+synchronized+and+geometrically+registered+mass+axotomy+in+a+three-compartmental+microfluidic+chip.">Wallerian-like degeneration of central neurons after synchronized and geometrically registered mass axotomy in a three-compartmental microfluidic chip.</a> Neurotox. Res. 19, 149-161 (2011).</li><li>Li, 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=Spatiotemporally+controlled+and+multifactor+involved+assay+of+neuronal+regeneration+after+chemical+injury+in+an+integrated+microfluidics.">Spatiotemporally controlled and multifactor involved assay of neuronal regeneration after chemical injury in an integrated microfluidics.</a> Anal. Chem. 84 (15), 6444-6453 (2012).</li><li>Kim, Y. T., Karthikeyan, K., Chirvi, S., Dave, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuro-optical+microfluidic+platform+to+study+injury+and+regeneration+of+single+axons.">Neuro-optical microfluidic platform to study injury and regeneration of single axons.</a> Lab Chip. 9, 2576-2581 (2009).</li><li>Hellman, A. N., 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=Examination+of+axonal+injury+and+regeneration+in+micropatterned+neuronal+culture+using+pulsed+laser+microbeam+dissection.">Examination of axonal injury and regeneration in micropatterned neuronal culture using pulsed laser microbeam dissection.</a> Lab Chip. 10 (16), 2083-2092 (2010).</li><li>Kunze, 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=Co-pathological+connected+primary+neurons+in+a+microfluidic+device+for+Alzheimer+studies.">Co-pathological connected primary neurons in a microfluidic device for Alzheimer studies.</a> Biotechnol. Bioeng. 108 (9), 2241-2245 (2011).</li><li>Liu, W. W., Goodhouse, J., Jeon, N. L., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+chamber+for+analysis+of+neuron-to-cell+spread+and+axonal+transport+of+an+alpha-herpesvirus.">A microfluidic chamber for analysis of neuron-to-cell spread and axonal transport of an alpha-herpesvirus.</a> PLoS One. 3 (6), (2008).</li><li>Markus, 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=Varicella-Zoster+virus+(VZV)+infection+of+neurons+derived+from+human+embryonic+stem+cells:+Direct+demonstration+of+axonal+infection,+transport+of+VZV,+and+productive+neuronal+infection.">Varicella-Zoster virus (VZV) infection of neurons derived from human embryonic stem cells: Direct demonstration of axonal infection, transport of VZV, and productive neuronal infection.</a> J. Virol. 85 (13), 6220-6233 (2011).</li><li>Dinh, N. 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=Microfluidic+construction+of+minimalistic+neuronal+co-cultures.">Microfluidic construction of minimalistic neuronal co-cultures.</a> Lab Chip. 13 (7), 1402-1412 (2013).</li><li>Tan, W. H., Takeuchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+trap-and-release+integrated+microfluidic+system+for+dynamic+microarray+applications.">A trap-and-release integrated microfluidic system for dynamic microarray applications.</a> Proc. Natl. Acad. Sci. USA. 104 (4), 1146-1151 (2007).</li><li>Frimat, J. -. 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=A+microfluidic+array+with+cellular+valving+for+single+cell+co-culture.">A microfluidic array with cellular valving for single cell co-culture.</a> Lab Chip. 11 (2), 231-237 (2011).</li><li>Di Carlo, D., Aghdam, N., Lee, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+enzyme+concentrations,+kinetics,+and+inhibition+analysis+using+high-density+hydrodynamic+cell+isolation+arrays.">Single-cell enzyme concentrations, kinetics, and inhibition analysis using high-density hydrodynamic cell isolation arrays.</a> Anal. Chem. 78 (14), 4925-4930 (2006).</li><li>Frimat, J. 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=The+network+formation+assay:+A+spatially+standardized+neurite+outgrowth+analytical+display+for+neurotoxicity+screening.">The network formation assay: A spatially standardized neurite outgrowth analytical display for neurotoxicity screening.</a> Lab Chip. 10, 701-709 (2010).</li><li>Hardelauf, 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=High+fidelity+neuronal+networks+formed+by+plasma+masking+with+a+bilayer+membrane:+analysis+of+neurodegenerative+and+neuroprotective+processes.">High fidelity neuronal networks formed by plasma masking with a bilayer membrane: analysis of neurodegenerative and neuroprotective processes.</a> Lab Chip. 11 (16), 2763-2771 (2011).</li><li>Whitesides, G. 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=Soft+lithography+in+biology+and+biochemistry.">Soft lithography in biology and biochemistry.</a> Annu. Rev. Biomed. Eng. 3, 335-373 (2001).</li><li>Haubert, K., Drier, T., Beebe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDMS+bonding+by+means+of+a+portable,+low-cost+corona+system.">PDMS bonding by means of a portable, low-cost corona system.</a> Lab Chip. 6, 1548-1549 (2006).</li><li>Frimat, J. 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=Plasma+stencilling+methods+for+cell+patterning.">Plasma stencilling methods for cell patterning.</a> Anal. Bioanal. Chem. 395 (3), 601-609 (2009).</li><li>Huang, N. 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=Poly(L-lysine)-g-poly(ethylene+glycol)+layers+on+metal+oxide+surfaces:+surface-analytical+characterization+and+resistance+to+serum+and+fibrinogen+adsorption.">Poly(L-lysine)-g-poly(ethylene glycol) layers on metal oxide surfaces: surface-analytical characterization and resistance to serum and fibrinogen adsorption.</a> Langmuir. 17, 489-498 (2001).</li><li>Li, N., Folch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+topographical+and+biochemical+cues+by+axons+during+growth+on+microfabricated+3-D+substrates.">Integration of topographical and biochemical cues by axons during growth on microfabricated 3-D substrates.</a> Exp. Cell Res. 311 (2), 307-316 (2005).</li><li>Huang, L. R., Cox, E. C., Austin, R. H., Sturm, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+particle+separation+through+deterministic+lateral+displacement.">Continuous particle separation through deterministic lateral displacement.</a> Science. 304 (5673), 987-990 (2004).</li><li>Yang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+nanoparticle+functionality+and+toxicity+on+the+central+nervous+system.">A review of nanoparticle functionality and toxicity on the central nervous system.</a> J. R. Soc. Interface. 7, (2010).</li><li>Brenneman, K. 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=Direct+olfactory+transport+of+inhaled+manganese+((MnCl2)-Mn-54)+to+the+rat+brain:+Toxicokinetic+investigations+in+a+unilateral+nasal+occlusion+model.">Direct olfactory transport of inhaled manganese ((MnCl2)-Mn-54) to the rat brain: Toxicokinetic investigations in a unilateral nasal occlusion model.</a> Toxicol. Appl. Pharm. 169 (3), 238-248 (2000).</li><li>Magal&#227;es, A. C., 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=Uptake+and+neuritic+transport+of+scrapie+prion+protein+coincident+with+infection+of+neuronal+cells.">Uptake and neuritic transport of scrapie prion protein coincident with infection of neuronal cells.</a> J. Neurosci. 25 (21), 5207-5216 (2005).</li><li>Diogenes, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+alpha-synuclein+oligomers+modulate+synaptic+transmission+and+impair+LTP+via+NMDA-receptor+activation.">Extracellular alpha-synuclein oligomers modulate synaptic transmission and impair LTP via NMDA-receptor activation.</a> J Neurosci. 32 (34), 11750-11762 (2012).</li><li>Egger, B., 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+imaging+of+primary+neural+cell+culture+from+Drosophila.">In vitro imaging of primary neural cell culture from Drosophila.</a> Nat. Protoc. 8 (5), 958-965 (2013).</li></ol>

作者宣称，他们有没有竞争的财务权益。

微流体组阵技术的先河，使精密单神经元的处理，建立简约的文化。再加上原位生物材料的构图方法，一种强大的方法，以使细胞形态与微结构，这些简约的文化有很高intercompartment连接水平与减少当地的纠缠。这些特征可以被用于室间传输的效率的研究，并且具有简单的设计修改必须分离单个轴突用于可视化和定量分析的潜力。例如，在下一代设计三叉戟结构可以与每个神经元长出1通道?...

<html>神经元组织是高度复杂的;即在空间中定义的图层和车厢，并用塑料连接通过细胞接触，特别是通过轴突和树突的副产物有序异质细胞的混合物。新技术必须赋予更大的自由度实验获得更深刻的见解和中央疾病，发展，健康功能绎机制。在Campenot室1,2和最近微细加工的实施例3,4可用于体外制备网络的神经元共培养物中以有选择地扰乱不同体细胞种群以及它们的轴突的副产物的能力。这些微流体装置已经例如被用于研究轴突变性和再生下述化学5,6或激光干切断6-8，τ病变9，病毒传播10,11，和mRNA定位于轴突4。 耳鼻喉科“&gt; 为了延长神经生物学家的覆盖范围，技术的发展需要编制简约神经元共培养。这使得神经网络的系统的单细胞​​和亚细胞精确调查的解缠结。为最小的细胞数目的要求打开来分析罕见的细胞类型，包括相关的帕金森氏病，从耳螺旋神经节，外周神经元的多巴胺能黑质细胞和干细胞的可能性。除此之外，蜂窝经济是相关的3R的主动权。使用这些微流体平台，大规模毒性帘或其他高吞吐量，数据丰富实验系列需要的动物的神经元现在可以考虑。 在本文中用于微流体装置的制作和使用的协议中说明。在原位 biomateri与微流控组合排列人的图案形成方法可用于使用最小细胞数目高度互连的神经元共培养物的登记。微流排列是基于差分流量的方法12-15，由此微陷阱被定位在流体回路（沿着与图1中的SEM图像示出）。路径0→1具有较低的流体阻力（R 2&gt; R 1）运送到神经元的微结构化孔的线性阵列-入口到神经突向外生长频道。由一个单细胞的陷阱占用本地阻碍了流动转移的流线对邻国陷阱诱捕随后的细胞。在阵列中的陷阱完全占用切换流体比（R 1&gt; R 2）的流线转移到蜿蜒路径（0→2），以产生动作的旁路模式以除去过量的神经元。 图1微流控电路。 A）差分电阻流体回路单神经元排列，与侧翼由轴突生长的渠道互联文化室，B，双层隔离的神经元共培养阵列与半月板钉扎micropillars权证）的SEM图像。有了这个设计，三叉形神经元诱捕结构被用于促进神经突起的副产物的颤动。图与传奇再现英国皇家化学学会（RSC）的许可12。 <a href="https://www.jove.com/files/ftp_upload/51389/51389fig1highres.jpg" target="_blank">点击这里查看大图。</a> 微图案神经网络对平面衬底的制备可以很容易地实现（对于为例<html>从我们的ES组，见Frimat 等 16海克等 17）。然而，在PDMS装置和与这些微米级的对准到微流体通道的要求包封生物活性材料图案构成了一个很大的技术挑战。在第3.1节中的协议为在芯片上，或原位制备的生物材料型态呈现。这些模式在漫长的文化时间表使神经元的登记和促进车厢之间的副产物。弯液面钉扎的微观结构是用来校准一个所谓水掩模与神经元的排列位点和神经突长出频道。水掩模保护粘附分子涂层中的等离子体处理，而暴露的表面被分解，以确定该生物材料的图案。此外，提供了用于细胞培养和对所需的不同共培养室的选择性治疗流体隔离协议。 ve_content“&gt;该协议旨在利用软光刻技术的人性化原则聚的复制二甲基硅氧烷（PDMS）微流控装置18。同样， 在原位生物材料的图案很简单，利用蒸发和表面张力的现象，只有需要一种廉价的手持式等离子体源的微流体回路有效方案小区负载和隔室进行这些操作的特定处理简单地分配材料到正确的底部端口和从上述吸移的问题。以这种方式，它的目的是提供对神经生物学家自由地在自己的实验室准备和使用微流体装置。

<table border="0" cellpadding="0" cellspacing="0" width="857">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">PDMS Sylgard 184</td>
			<td style="width:163px;">Dow Corning</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PDMS Elastosil RT 601</td>
			<td style="width:163px;">Wacker</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Coverslips</td>
			<td style="width:163px;">VWR</td>
			<td>630-1590</td>
			<td>130-160 mm thick</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">3 mm Biopsy Punches</td>
			<td style="width:163px;">Kai Medical</td>
			<td style="width:143px;"></td>
			<td>Handle with care - extremely sharp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tygon Tubing</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:143px;">S-50-HL</td>
			<td>1.65 mm ID; 3.35 mm OD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mL Syringe (Inkjekt and Omnifix)</td>
			<td style="width:163px;">Braun</td>
			<td style="width:143px;">6064204</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">4-Way Tubing Connector</td>
			<td style="width:163px;">VWR or Fisher Scientific</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Flow Regulator</td>
			<td style="width:163px;">Harvard Apparatus</td>
			<td style="width:143px;">722645</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">0.5 mm Pins</td>
			<td style="width:163px;">Dressmaking Departments</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">PLL-g-PEG</td>
			<td style="width:163px;">SuSoS</td>
			<td style="width:143px;">PLL(20)-g[3.5]-PEG(5)</td>
			<td>Stability in storage can be an issue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">poly-D-lysine</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">P6407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">poly-lysine-FITC</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">P3069</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">poly-ornithine</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">P4957</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">fibronectin</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">F2006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">laminin</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">L2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">PBS</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">P4417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Inverted Fluorescent Microscope</td>
			<td style="width:163px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">Example Aspiration Pump</td>
			<td style="width:163px;">KNF Neuberger, Laboport</td>
			<td style="width:143px;">N811KVP</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">Hand Held Corona Discharge Device</td>
			<td style="width:163px;">Leybold-Heraeus, USA</td>
			<td style="width:143px;">VP23</td>
			<td>May not comply with your country&#39;s safety standards</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Femto Plasma Oven</td>
			<td style="width:163px;">Diener Electronic</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Vacuum Dessicator or Centrifuge</td>
			<td style="width:163px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of neuronal co-cultures with single cell precision

该Campenot室的微流控实施方案已经从神经科学界引起了极大的兴趣。这些互连的共培养平台，可用于调查的各种问题，跨越发育和功能性神经生物学感染和疾病传播。然而，传统的系统需要显著蜂窝输入（几千每隔室），不足用于研究低丰度细胞，如初级多巴胺能黑质，螺旋神经节，和果蝇黑腹果蝇的神经元，以及不切实际的高通量实验。密集的文化也备受本地纠缠，很少有副产物（&lt;10％）互连的两种文化。在本文中简单的微流体和图案化的协议，描述了解决这些问题：（ⅰ）一种微流体的单神经元排列方法，以及（ii）水掩蔽方法为等离子体图案形成生物材料涂层来REGISTER神经元，促进车厢之间的产物。简约神经元共培养物制备了高水平（&gt; 85％）intercompartment连接和可用于高通量神经生物学实验用单细胞精度。

水屏蔽攻击表面张力。在空气 - 液体界面的压力耐受性成反比带的曲率[半径<img fo:content-width="1in" src="files/ftp_upload/51389/51389eq1.jpg" width="113" /> ，生产高度稳定的接口在微尺寸。该micropillars有效锚定水面膜到位。补水面膜的形成是从微端口驱动的蒸发，随着速度提高加热。从低倍（4X - 10X）使用热显微镜照明，水面膜通常是建立在≤5分钟。蒸发也导致了水掩模的最终倒塌。增加水面膜的一辈子〜15?...

Watch this Scientific Journal Video about Preparation of Neuronal Co-cultures with Single Cell Precision at JoVE.com

Preparation of Neuronal Co-cultures with Single Cell Precision

协议的单神经元微排列和水掩蔽生物材料涂层在芯片血浆图形描述。高度互联共培养可以使用最小的单元格输入做好准备。

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 ...

preparation-of-neuronal-co-cultures-with-single-cell-precision

Research

JoVE Journal

Neuroscience

13.6K 次观看.  ISAS. 协议的单神经元微排列和水掩蔽生物材料涂层在芯片血浆图形描述。高度互联共培养可以使用最小的单元格输入做好准备。 

视频: Preparation of Neuronal Co-cultures with Single Cell Precision

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

丰富的少突胶质细胞培养和Myelinating产后小鼠组织联合培养的少突胶质细胞/神经元的推导

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

科研

神经科学

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

单细胞的多巴胺释放，同时电压钳和电流分析法测量

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

阈上的神经活动的单细胞和单穗的分辨率的光学的方法记录

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

同时前和突触后电生理记录<em&gt;爪蟾</em神经肌肉文化

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

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 ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

调查器官脊髓联合培养物中生长多电极阵列功能再生

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

在子宫内电途径研究神经元亚群的兴奋性和单细胞连接

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which macrophages are induced to produce conditioned medium (CM) that promotes neurite outgrowth. Adult dorsal root ganglion (DRG) neurons are acutely dissociated and plated. After the neurons are stably attached, peritoneal macrophages are co-cultured on a cell culture insert overlaid on the same well. Dibutyryl cyclic AMP (db-cAMP) is applied to the co-cultures for 24 h, after which the cell culture insert containing the macrophages is moved to another well to collect CM for 72 h. The CM from the co-cultures treated with db-cAMP, when applied to a separate adult DRG neuron culture, exhibits robust neurite outgrowth activity. The CM obtained from the db-cAMP-treated cultures consisting of single cell type alone, either DRG neuron or peritoneal macrophage, did not exhibit neurite outgrowth activity. This indicates that the interaction between neurons and macrophages is indispensable for the activation of macrophages secreting molecular factors with neurite outgrowth activity into CM. Thus, our co-culture paradigm will also be useful to study intercellular signaling in the neuron-macrophage interaction to stimulate the macrophages to be endowed with a pro-regenerative phenotype.

The current protocol presents experimental procedures to stimulate cultured macrophages to be endowed with capacity to release molecular factors that promote neurite outgrowth. Treatment of cAMP to the neuron-macrophage co-cultures induces the macrophages to produce conditioned medium that possesses strong neurite outgrowth activity.

神经元-巨噬细胞共培养活化巨噬细胞分泌突起活性的分子因子

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which ...