这项工作由国家科学基金会授予的CMMI-1300007资助。我们要感谢以前的实验室成员，他们帮助设计这些协议，并在乔治梅森大学维持文化五年以上:Dr. J. J. Pancrazio，Hamid Charkhkar博士，Gretchen Knaack博士，Franz Hamilton博士，Michael Maquera博士和Robert Graham。

<ol>
	<li>Charkhkar, H., Frewin, 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=Use+of+cortical+neuronal+networks+for+in+vitro+material+biocompatibility+testing.">Use of cortical neuronal networks for in vitro material biocompatibility testing.</a> Biosens Bioelectron. 53, 316-323 (2014).</li><li>Bologna, L. L., Nieus, T., Tedesco, M., Chiappalone, M., Benfenati, F., 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=Low-frequency+stimulation+enhances+burst+activity+in+cortical+cultures+during+development.">Low-frequency stimulation enhances burst activity in cortical cultures during development.</a> Neuroscience. 165 (3), 692-704 (2010).</li><li>Fr&#246;hlich, F., McCormick, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+electric+fields+may+guide+neocortical+network+activity.">Endogenous electric fields may guide neocortical network activity.</a> Neuron. 67 (1), 129-143 (2010).</li><li>Anastassiou, C. A., Perin, R., Markram, H., Koch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ephaptic+coupling+of+cortical+neurons.">Ephaptic coupling of cortical neurons.</a> Nature Neurosci. 14 (2), 217-223 (2011).</li><li>Ozen, S., Sirota, 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=Transcranial+electric+stimulation+entrains+cortical+neuronal+populations+in+rats.">Transcranial electric stimulation entrains cortical neuronal populations in rats.</a> J. Neurosci. 30 (34), 11476-11485 (2010).</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 (3), 680-688 (2005).</li><li>Shahaf, G., 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=Learning+in+networks+of+cortical+neurons.">Learning in networks of cortical neurons.</a> J. Neurosci. 21 (22), 8782-8788 (2001).</li><li>Marom, S., Shahaf, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development,+learning+and+memory+in+large+random+networks+of+cortical+neurons:+lessons+beyond+anatomy.">Development, learning and memory in large random networks of cortical neurons: lessons beyond anatomy.</a> Q Rev Biophys. 35 (1), 63-87 (2002).</li><li>Marom, S., Eytan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+in+ex-vivo+developing+networks+of+cortical+neurons.">Learning in ex-vivo developing networks of cortical neurons.</a> Prog Brain Res. 147, 189-199 (2005).</li><li>Li, Y., Zhou, W., Li, X., Zeng, S., Luo, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+learning+in+cultured+neuronal+networks+with+antagonists+of+glutamate+receptors.">Dynamics of learning in cultured neuronal networks with antagonists of glutamate receptors.</a> Biophys. J. 93 (12), 4151-4158 (2007).</li><li>Li, Y., Zhou, W., Li, X., Zeng, S., Liu, M., Luo, Q. <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+synchronized+bursts+in+cultured+hippocampal+neuronal+networks+with+learning+training+on+microelectrode+arrays.">Characterization of synchronized bursts in cultured hippocampal neuronal networks with learning training on microelectrode arrays.</a> Biosens Bioelectron. 22 (12), 2976-2982 (2007).</li><li>Chiappalone, M., Massobrio, P., 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+plasticity+in+cortical+assemblies.">Network plasticity in cortical assemblies.</a> Eur. J. Neurosci. 28 (1), 221-237 (2008).</li><li>le Feber, J., Stegenga, J., Rutten, W. L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+slow+electrical+stimuli+to+achieve+learning+in+cultured+networks+of+rat+cortical+neurons.">The effect of slow electrical stimuli to achieve learning in cultured networks of rat cortical neurons.</a> PloS one. 5 (1), 8871 (2010).</li><li>Ruaro, M. E., Bonifazi, P., Torre, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+the+neurocomputer:+image+processing+and+pattern+recognition+with+neuronal+cultures.">Toward the neurocomputer: image processing and pattern recognition with neuronal cultures.</a> IEEE Trans Biomed Eng. 52 (3), 371-383 (2005).</li><li>Hamilton, F., Graham, 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=Time-Dependent+Increase+in+Network+Response+to+Stimulation.">Time-Dependent Increase in Network Response to Stimulation.</a> PloS one. 10 (11), 0142399 (2015).</li><li>. Guidelines for Euthanasia of Rodents Using Carbon Dioxide Available from: <a target="_blank" href="https://oacu.oir.nih.gov/sites/default/files/uploads/arac-guidelines/rodent_euthanasia_adult.pdf">https://oacu.oir.nih.gov/sites/default/files/uploads/arac-guidelines/rodent_euthanasia_adult.pdf</a> (2016)</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 Cells: Business Source. , (1998).</li><li>Gullo, F., Maffezzoli, A., Dossi, E., Wanke, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-latency+cross-and+autocorrelation+identify+clusters+of+interacting+cortical+neurons+recorded+from+multi-electrode+array.">Short-latency cross-and autocorrelation identify clusters of interacting cortical neurons recorded from multi-electrode array.</a> J. Neurosci. Methods. 181, 186-198 (2009).</li><li>Eytan, D., Brenner, N., 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=Selective+Adaptation+in+Networks+of+Cortical+Neurons.">Selective Adaptation in Networks of Cortical Neurons.</a> J. Neurosci. 23 (28), (2003).</li><li>Bonifazi, P., Ruaro, M. E., Torre, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+properties+of+information+processing+in+neuronal+networks.">Statistical properties of information processing in neuronal networks.</a> Eur. J. Neurosci. 22 (11), 2953-2964 (2005).</li></ol>

作者没有什么可以披露的。

本协议中概述的步骤提供了足够的细节，让初学者在多边环境协定中打造自己的神经元文化，并记录网络活动。该方案将有助于确保培养物正确粘附，在电极阵列上形成细胞层的地毯层，并保持健康和无污染的数月。 尽管最好遵守协议的所有部分，但整个过程中仍然存在对成功结果至关重要的步骤。在整个过程中使用无菌技术是防止培养物被污染的必要条件。如方案中所述，新的MEA必须是亲水的，否则将导致细胞粘附不良。在分离过程中避免刺激吸气和气泡形成会减少受损细胞的数量，并将导致更高和更健康的产量。第一次从DMEM 5/5切换到DMEM +喂养也很重要。 DMEM 5/5含有马血清，如果连续使用，会导致胶质细胞占主导地位，并导致差的神经元活性，尽管培养物将呈现健康状态。按照计划喂养文化，使其保持在适当的孵化条件下也是至关重要的。 多孔介质上的电镀细胞培养涉及许多可能导致不太理想结果的变量。虽然目标是细胞的完美"地毯"，但是未能解决上述关键步骤将导致细胞成熟或污染不良。细胞粘附不良与细胞成熟不同也是一个问题。这可以由几个因素引起，包括在电镀之前的MEA制备不良或使用旧介质。如果含有稳定形式的L-谷氨酰胺和用于神经细胞培养的无血清补充剂的老培养基（参见材料 表），细胞最初粘附，然后在约两周后漂浮。如果细菌污染是一个持续存在的问题，可以向培养基中加入抗生素，如氨苄青霉素或pen-strep。还有可用于治疗真菌污染的杀真菌剂。这些是可能影响文化结果的一些更常见的变量。还有很多其他的时间和经验才会遇到。 与使用玻璃微电极相比，该技术非常适用于研究网络动力学和药理学反应。它能够使用许多不同的时空刺激模式，并允许立即从多个区域记录神经元反应。以前的团体使用类似于这里描述的协议来证明有趣的结果18 。由于文化持续数周或数月，同样的文化可以重复使用，这种技术也是一样在同一网络上随着时间推移进行多次实验。 然而，这种技术有局限性。多边环境协定是非侵入性的。因此，它们只能记录细胞外活性，而不是用移液管进行贴片钳或细胞内记录。此外，由于阵列中的每个电极被几个细胞覆盖，所以不可能解决单个神经元的活性。相反，因为这些是体外培养，它们不能完全复制大脑网络的结构性质。此外，活性只能一次记录少于30分钟，没有一些机制为细胞提供CO 2气氛以保持其pH平衡。 一旦掌握了这种技术，就可以探索有或没有电刺激的药理学操作。探索神经网络学习和记忆形成的新方案也可以设计和测试，以及p海马或脊髓网络协议。以前已经公布了刺激和训练网络的方案，其中一些进一步发展成体内协议，如Eytan 等人提出的"选择性适应" 。 19 。测试了几种方案。然而，仅在Ruaro 2005年提出的破伤风手术修改的结果在这里提出了14,20。

微电极阵列（MEA）可用于研究药物毒性，下一代个性化药物的设计范例，以及神经元培养中的网络动力学研究1 。与更传统的方法相反，例如仅能记录单个细胞的活性的片段钳位，或者用玻璃移液管进行现场记录，其可以记录来自单个位点的电极周围的神经元的细胞外反应--MOA可以同时在细胞培养中从多个位点记录，而不需要分别放置每个电极的艰巨任务。这允许研究在该文化中形成网络的细胞群之间的动态相互作用。此外，电刺激对网络发射模式2,3,4,5和网络控制6的影响/ sup&gt;在神经元培养物中已经有很好的记录，并且可以在相同的实验装置中容易地应用许多电刺激和对照的配置，从而允许探索广泛的时空动力学。 这些体外研究感兴趣的关键动力之一是培养的网络显示表示学习的性质7,8,9,10,11,12,13。 Peixoto实验室以前研究了高频训练信号的影响，如Ruaro et al。 14 ，在微电极阵列15上铺板的小鼠神经网络上。在这些实验中，网络显示响应跟随性增加g电刺激引起的训练。增加的反应被认为是通过刺激识别的一种学习形式，其中网络在应用特定刺激（ 即培训）方案之后以一致的方式响应于刺激的改变。 该协议演示如何培养MEA上的神经元细胞，成功记录超过95％的电镀盘，建立培养网络以响应刺激模式，排序单一单位活动，绘制直方图并解释结果的方案这样的实验。证明使用专有系统（参见材料表 ）来刺激和记录神经元培养物，以及软件包的应用（参见材料表 ）来排序神经元单位。定制设计的图形用户界面（参见表格材料 ）用于可视化后期时间延迟时间直方图，突发间间隔和突发持续时间，以及将训练协议之前和之后的细胞响应与刺激进行比较。

<table><tbody><tr><td>Ascorbic Acid</td><td>Sigma Aldrich</td><td>A4403</td><td>Make 4 mg/mL aliquots</td></tr><tr><td>B-27</td><td>Invitrogen</td><td>17504-044</td><td>Serum-free supplement for neural cell culture. Make 1 mL aliquots and freeze</td></tr><tr><td>Beakers - glass - 100 mL</td><td>VWR</td><td>13912-160</td><td></td></tr><tr><td>Beakers - glass - 500 mL</td><td>VWR</td><td>10754-956</td><td></td></tr><tr><td>Blunt-tipped thumb forceps</td><td>World Precision Instruments</td><td>500365-G</td><td></td></tr><tr><td>Cryogenic vial - sterile, 2 mL</td><td>Fisher Scientific</td><td>10-500-26</td><td></td></tr><tr><td>Cryogenic vial - sterile, 5 mL</td><td>Fisher Scientific</td><td>10-500-27</td><td></td></tr><tr><td>DMEM with Glutamax</td><td>Gibco Life Technology</td><td>10569-010</td><td>Cell culture media that contains a stabilized form of L-glutamine</td></tr><tr><td>DNase</td><td>Worthington Biochemical</td><td>LK003172</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>04-355-451</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>ATCC</td><td>30-2030</td><td></td></tr><tr><td>Fine-tipped thumb forceps</td><td>World Precision Instruments</td><td>501324-G</td><td></td></tr><tr><td>Glass Pipets</td><td>VWR</td><td>14673-010</td><td></td></tr><tr><td>GlutaMAX -- I (100X)</td><td>Gibco Life Technology</td><td>35050-061</td><td>A stabilized form of L-glutamine used as a suplement</td></tr><tr><td>Hemocytometer chip</td><td>Fisher Scientific</td><td>22-600-101</td><td></td></tr><tr><td>Hibernate EB complete</td><td>BrainBits</td><td>D00118</td><td>Ambient CO&lt;sup&gt;2&lt;/sup&gt; cell storage media</td></tr><tr><td>Horse Serum</td><td>Atlanta Biologicals</td><td>S12195H</td><td></td></tr><tr><td>Laminin</td><td>Sigma Aldrich</td><td>L2020-1MG</td><td></td></tr><tr><td>MCS filter amplifier</td><td>MultiChannel System</td><td>FA60SBC</td><td></td></tr><tr><td>MCS headstage/pre-amplifier</td><td>MultiChannel System</td><td>MEA1060-INV</td><td></td></tr><tr><td>MCS microelectrode array</td><td>MultiChannel System</td><td>60MEA200/10iR-ITO</td><td></td></tr><tr><td>MCS power supply</td><td>MultiChannel System</td><td>PS20W</td><td></td></tr><tr><td>MCS signal divider</td><td>MultiChannel System</td><td>SDMEA</td><td></td></tr><tr><td>MCS stimulus generator</td><td>MultiChannel System</td><td>STG4002</td><td></td></tr><tr><td>MCS temperature controller</td><td>MultiChannel System</td><td>TC02</td><td></td></tr><tr><td>Media storage bottle - glass 500 mL</td><td>VWR</td><td>10754-818</td><td>Autoclave</td></tr><tr><td>Microcentrifuge tubes - 0.4 mL</td><td>Thermo Fisher</td><td>3485</td><td>Autoclave</td></tr><tr><td>Microcentrifuge tubes - 2 mL</td><td>Thermo Fisher</td><td>3434ECONO</td><td>Autoclave</td></tr><tr><td>Micropipette tips - 10 &amp;mu;L</td><td>VWR</td><td>37001-166</td><td>Autoclave</td></tr><tr><td>Micropipette tips - 1,000 &amp;mu;L&amp;nbsp;</td><td>VWR</td><td>13503-464</td><td>Autoclave</td></tr><tr><td>Micropipette tips - 200 &amp;mu;L&amp;nbsp;</td><td>VWR</td><td>37001-596</td><td>Autoclave</td></tr><tr><td>Micropipetter - 10 &amp;mu;L&amp;nbsp;</td><td>Thermo Fisher</td><td>4641170N</td><td></td></tr><tr><td>Micropipetter - 1,000 &amp;mu;L&amp;nbsp;</td><td>Thermo Fisher</td><td>4641210N</td><td></td></tr><tr><td>Micropipetter - 200 &amp;mu;L&amp;nbsp;</td><td>Thermo Fisher</td><td>4641230N</td><td></td></tr><tr><td>Papain - 0.22 Filtered</td><td>Worthington Biochemical</td><td>LK003178</td><td></td></tr><tr><td>Penicillin-Streptomycin (Pen Strep)</td><td>Thermo Fisher</td><td>15070063</td><td></td></tr><tr><td>Petri dishes -100 mm disposable</td><td>Fisher Scientific</td><td>08-757-100D</td><td></td></tr><tr><td>Petri dishes -100 mm glass</td><td>VWR</td><td>10754-788</td><td></td></tr><tr><td>Petri dishes -35 mm&amp;nbsp;</td><td>Fisher Scientific</td><td>08-757-100A</td><td></td></tr><tr><td>Phosphate buffer saline (PBS) (1x)</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>Plasma Cleaning System</td><td>Plasma Etch, Inc.</td><td>PE-50</td><td></td></tr><tr><td>Poly-D-lysine hydrobromide (PDL)</td><td>Sigma Aldrich</td><td>P6407-5MG</td><td></td></tr><tr><td>Polyethylene conical (centrifuge) tube -15 mL</td><td>Fisher Scientific</td><td>05-527-90</td><td></td></tr><tr><td>Polypropylene conical (centrifuge) tube -50 mL</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Procedure masks</td><td>Imco</td><td>1530-imc</td><td></td></tr><tr><td>Pyruvate</td><td>Sigma Aldrich</td><td>P4562-5G</td><td></td></tr><tr><td>Scalpel blades</td><td>World Precision Instruments</td><td>&amp;nbsp;500237-G</td><td></td></tr><tr><td>Scissors - iris</td><td>World Precision Instruments</td><td>14111-G</td><td></td></tr><tr><td>Scissors - large</td><td>World Precision Instruments</td><td>14214</td><td></td></tr><tr><td>Scissors - small surgical</td><td>World Precision Instruments</td><td>&amp;nbsp;501733-G</td><td></td></tr><tr><td>Serological pipette - sterile, 1 mL&amp;nbsp;</td><td>VWR</td><td>89130-892</td><td></td></tr><tr><td>Serological pipette - sterile, 2 mL&amp;nbsp;</td><td>VWR</td><td>89130-894</td><td></td></tr><tr><td>Serological pipette - sterile, 25 mL&amp;nbsp;</td><td>VWR</td><td>89130-900</td><td></td></tr><tr><td>Serological pipette - sterile, 50 mL&amp;nbsp;</td><td>VWR</td><td>89130-902</td><td></td></tr><tr><td>Software: Matlab GUI</td><td>Peixoto Lab</td><td>npeixoto@gmu.edu</td><td>Used to analyze .mat files.&amp;nbsp; Available for free upon request.&amp;nbsp; Contact npeixoto@gmu.edu to request a copy.</td></tr><tr><td>Software: MCS MC_Rack</td><td>MultiChannel System</td><td>N/A</td><td>Used for data acquisition</td></tr><tr><td>Software: NeuroExplorer</td><td>Plexon&amp;nbsp;</td><td>http://www.plexon.com/products/neuroexplorer</td><td>Used to convert .nex files to .mat</td></tr><tr><td>Software: Offline Sorter</td><td>Plexon&amp;nbsp;</td><td>http://www.plexon.com/products/offline-sorter</td><td>Used to sort neural spikes and convert data files to .plx and then to .nex</td></tr><tr><td>Software Manual: MCS MC_Rack</td><td>MultiChannel System</td><td></td><td>https://www.multichannelsystems.com/sites/multichannelsystems.com/files/documents/manuals/MC_Rack_Manual.pdf</td></tr><tr><td>Software Manual: NeuroExplorer</td><td>Plexon&amp;nbsp;</td><td></td><td>https://www.neuroexplorer.com/downloads/Nex3Manual.pdf</td></tr><tr><td>Software Manual: Offline Sorter</td><td>Plexon&amp;nbsp;</td><td></td><td>www.plexon.com/system/files/downloads/Offline%20Sorter%20v2.8%20Manual.pdf</td></tr><tr><td>Spatula - small double-ended</td><td>World Precision Instruments</td><td>&amp;nbsp;503440</td><td></td></tr><tr><td>Stericup 0.22 &amp;micro;m pore filter - 250 mL</td><td>Millipore</td><td>SCGVU02RE</td><td></td></tr><tr><td>Transfer pipette - large bore&amp;nbsp;</td><td>Thermo Fisher</td><td>335-1S</td><td></td></tr><tr><td>Transfer pipette - small bore&amp;nbsp;</td><td>Thermo Fisher</td><td>242-1S</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma Aldrich</td><td>T8154-20ML</td><td></td></tr><tr><td>Whatman filter paper</td><td>Whatman</td><td>1442 150</td><td>Cut into 8 pie wedges and autoclave in a glass Petri dish</td></tr></tbody></table>

time-dependent increase in the network response to the stimulation of neuronal cell cultures on micro-electrode arrays

微电极阵列（MEA）可用于研究药物毒性，为下一代个性化医学设计范例，研究神经元培养中的网络动力学。与更传统的方法（例如仅能记录单个单元的活动的补丁夹）相反，MEA可以从网络中的多个站点同时记录，而不需要单独放置每个电极的艰巨任务。此外，可以在相同的实验装置中容易地应用许多控制和刺激配置，从而允许探索广泛的动力学。这些体外研究感兴趣的关键动力之一是培养的网络显示表征学习的属性的程度。在MEA上培养的小鼠神经元细胞显示由电刺激诱导的训练后的反应增加。该协议演示了如何在MEA上培养神经元细胞;成功地从超过95％的电镀盘中绳索;建立协议来训练网络来应对刺激模式;并对这些实验的结果进行排序，绘制和解释。证明了使用专有系统来刺激和记录神经元培养。软件包也用于排序神经元单位。定制设计的图形用户界面用于可视化刺激后时间直方图，脉冲间间隔和脉冲串持续时间，以及将训练协议之前和之后的细胞响应与刺激进行比较。最后，讨论了这项研究工作的代表性成果和未来发展方向。

Watch this Scientific Journal Video about Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays at JoVE.com

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

在多电极阵列上培养的小鼠神经元细胞在电刺激后显示出响应的增加。该协议演示了如何培养神经元，如何记录活动，以及如何建立协议来训练网络来应对刺激模式。

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 ...

time-dependent-increase-network-response-to-stimulation-neuronal-cell

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.8K 次观看.  George Mason University. 在多电极阵列上培养的小鼠神经元细胞在电刺激后显示出响应的增加。该协议演示了如何培养神经元，如何记录活动，以及如何建立协议来训练网络来应对刺激模式。 

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

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

Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday products. They are classified as endocrine disrupting compounds (EDC) with estradiol-like properties. Long-term exposure to EDCs, even at low doses, has been linked with various health defects including cancer, behavioral disorders, and infertility, with greater vulnerability during early developmental periods. To study the effects of BPA on the development of neuronal function, we used an in vitro neuronal network derived from the early chick embryonic brain as a model. We found that exposure to BPA affected the development of network activity, specifically spiking activity and synchronization. A change in network activity is the crucial link between the molecular target of a drug or compound and its effect on behavioral outcome. Multi-electrode arrays are increasingly becoming useful tools to study the effects of drugs on network activity in vitro. There are several systems available in the market and, although there are variations in the number of electrodes, the type and quality of the electrode array and the analysis software, the basic underlying principles, and the data obtained is the same across the different systems. Although currently limited to analysis of two-dimensional in vitro cultures, these MEA systems are being improved to enable in vivo network activity in brain slices. Here, we provide a detailed protocol for embryonic exposure and recording neuronal network activity and synchrony, along with representative results.

Exposure to environmental toxins can acutely impact developing embryos. Endocrine disrupting chemicals such as bisphenols are known to adversely affect the nervous system. Here we describe a protocol using an in vitro vertebrate (chick embryo) neuronal network model to study the functional impact of toxin exposure on early embryos.

应用多电极阵列评价内分泌干扰物对脊椎动物神经网络功能发育的影响

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday ...

发育生物学

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

Closed-loop Neuro-robotic Experiments to Test Computational Properties of Neuronal Networks

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation of a given stimulus might be dependent on context and history. If this is actually the case, bi-directional interactions between the brain (or if need be a reduced model of it) and sensory-motor system can shed a light on how encoding and decoding of information is performed. Here an experimental system is introduced and described in which the activity of a neuronal element (i.e., a network of neurons extracted from embryonic mammalian hippocampi) is given context and used to control the movement of an artificial agent, while environmental information is fed back to the culture as a sequence of electrical stimuli. This architecture allows a quick selection of diverse encoding, decoding, and learning algorithms to test different hypotheses on the computational properties of neuronal networks.

In this paper, an experimental framework to perform closed-loop experiments is presented, in which information processing (i.e., coding and decoding) and learning of neuronal assemblies are studied during the continuous interaction with a robotic body.

闭环神经机器人实验来检验神经网络的计算性能

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation ...

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 ...

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

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system.
In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

使用电动和一维和二维文化磁场神经元的外部激励

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

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

微电极阵列接口微流体研究神经元通信和轴向信号传播

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

生物工程

Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an increasingly detailed view of the circuits underpinning spinal pain processing. However, the effects of these connections for broader network activity in the DH remain less well understood because most studies focus on the activity of single neurons and small microcircuits. Alternatively, the use of microelectrode arrays (MEAs), which can monitor electrical activity across many cells, provides high spatial and temporal resolution of neural activity. Here, the use of MEAs with mouse spinal cord slices to study DH activity induced by chemically stimulating DH circuits with 4-aminopyridine (4-AP) is described. The resulting rhythmic activity is restricted to the superficial DH, stable over time, blocked by tetrodotoxin, and can be investigated in different slice orientations. Together, this preparation provides a platform to investigate DH circuit activity in tissue from na&#239;ve animals, animal models of chronic pain, and mice with genetically altered nociceptive function. Furthermore, MEA recordings in 4-AP-stimulated spinal cord slices can be used as a rapid screening tool to assess the capacity of novel antinociceptive compounds to disrupt activity in the spinal cord DH.

The combined use of microelectrode array technology and 4-aminopyridine-induced chemical stimulation for investigating network-level nociceptive activity in the spinal cord dorsal horn is outlined.

使用微电极阵列记录脊髓伤害感受回路中的网络活动

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an ...

3D 打印在微型神经元细胞培养设备中的应用

Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, and realistic synaptic connectivity are missing. The latter limits the study of structure and function in the context of compartmentalization and diminishes the significance of cultures in neuroscience. Approximating ex vivo the structured anatomical arrangement of synaptic connectivity is not trivial, despite being key for the emergence of rhythms, synaptic plasticity, and ultimately, brain pathophysiology. Here, two-photon polymerization (2PP) is employed as a 3D printing technique, enabling the rapid fabrication of polymeric cell culture devices using polydimethyl-siloxane (PDMS) at the micrometer scale. Compared to conventional replica molding techniques based on microphotolitography, 2PP micro-scale printing enables rapid and affordable turnaround of prototypes. This protocol illustrates the design and fabrication of PDMS-based microfluidic devices aimed at culturing modular neuronal networks. As a proof-of-principle, a two-chamber device is presented to physically constrain connectivity. Specifically, an asymmetric axonal outgrowth during ex vivo development is favored and allowed to be directed from one chamber to the other. In order to probe the functional consequences of unidirectional synaptic interactions, commercial microelectrode arrays are chosen to monitor the bioelectrical activity of interconnected neuronal modules. Here, methods to 1) fabricate molds with micrometer precision and 2) perform in vitro multisite extracellular recordings in rat cortical neuronal cultures are illustrated. By decreasing costs and future widespread accessibility of 2PP 3D-printing, this method will become more and more relevant across research labs worldwide. Especially in neurotechnology and high-throughput neural data recording, the ease and rapidity of prototyping simplified in vitro models will improve experimental control and theoretical understanding of in vivo large-scale neural systems.

作者聚焦:用于分析大脑功能的模块化神经元网络

3D printing at the micrometer scale enables rapid prototyping of polymeric devices for neuronal cell cultures. As a proof-of-principle, structural connections between neurons were constrained by creating barriers and channels influencing neurite outgrowth, while the functional consequences of such manipulation were observed by extracellular electrophysiology.

微尺度神经元细胞培养装置的双光子聚合3D打印

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, ...

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

摘要

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此视频中的章节

器官型培养的神经元雪崩多电极阵列记录

应用多电极阵列评价内分泌干扰物对脊椎动物神经网络功能发育的影响

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）

闭环神经机器人实验来检验神经网络的计算性能

接口3D工程神经细胞微电极阵列:一种创新

使用电动和一维和二维文化磁场神经元的外部激励

微电极阵列接口微流体研究神经元通信和轴向信号传播

使用微电极阵列记录脊髓伤害感受回路中的网络活动

微尺度神经元细胞培养装置的双光子聚合3D打印

微尺度神经元细胞培养装置的双光子聚合3D打印