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>
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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 border="0" cellpadding="0" cellspacing="0" width="1135">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Ascorbic Acid</td>
			<td style="width:204px;">Sigma Aldrich</td>
			<td style="width:155px;">A4403</td>
			<td style="width:356px;">Make 4mg/mL aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">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 height="21">
			<td height="21" style="height:21px;width:420px;">Beakers - glass - 100 mL</td>
			<td>VWR</td>
			<td>13912-160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Beakers - glass - 500 mL</td>
			<td>VWR</td>
			<td>10754-956</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Blunt-tipped thumb forceps</td>
			<td>World Precision Instruments</td>
			<td>500365-G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Cryogenic vial - sterile, 2 mL</td>
			<td>Fisher Scientific</td>
			<td>10-500-26</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Cryogenic vial - sterile, 5 mL</td>
			<td>Fisher Scientific</td>
			<td>10-500-27</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">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 height="21">
			<td height="21" style="height:21px;width:420px;">DNase</td>
			<td>Worthington Biochemical</td>
			<td>LK003172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Ethanol</td>
			<td>Fisher Scientific</td>
			<td>04-355-451</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Fetal bovine serum (FBS)</td>
			<td>ATCC</td>
			<td>30-2030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Fine-tipped thumb forceps</td>
			<td>World Precision Instruments</td>
			<td style="width:155px;">501324-G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Glass Pipets</td>
			<td>VWR</td>
			<td>14673-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">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 height="21">
			<td height="21" style="height:21px;width:420px;">Hemocytometer chip</td>
			<td>Fisher Scientific</td>
			<td>22-600-101</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:420px;">Hibernate EB complete</td>
			<td>BrainBits</td>
			<td>D00118</td>
			<td>Ambient CO2 cell storage media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Horse Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S12195H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Laminin</td>
			<td>Sigma Aldrich</td>
			<td>L2020-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">MCS filter amplifier</td>
			<td>MultiChannel System</td>
			<td>FA60SBC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">MCS headstage/pre-amplifier</td>
			<td>MultiChannel System</td>
			<td>MEA1060-INV</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:420px;">MCS microelectrode array</td>
			<td>MultiChannel System</td>
			<td>60MEA200/10iR-ITO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">MCS power supply</td>
			<td>MultiChannel System</td>
			<td>PS20W</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">MCS signal divider</td>
			<td>MultiChannel System</td>
			<td>SDMEA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">MCS stimulus generator</td>
			<td>MultiChannel System</td>
			<td>STG4002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">MCS temperature controller</td>
			<td>MultiChannel System</td>
			<td>TC02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Media storage bottle - glass 500 mL</td>
			<td>VWR</td>
			<td>10754-818</td>
			<td>Autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Microcentrifuge tubes - 0.4 mL</td>
			<td>Thermo Fisher</td>
			<td>3485</td>
			<td>Autoclave</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:420px;">Microcentrifuge tubes - 2 mL</td>
			<td>Thermo Fisher</td>
			<td>3434ECONO</td>
			<td>Autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Micropipette tips - 10 uL&#160;</td>
			<td>VWR</td>
			<td>37001-166</td>
			<td>Autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Micropipette tips - 1000 uL&#160;</td>
			<td>VWR</td>
			<td>13503-464</td>
			<td>Autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Micropipette tips - 200 uL&#160;</td>
			<td>VWR</td>
			<td>37001-596</td>
			<td>Autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Micropipetter - 10 uL&#160;</td>
			<td>Thermo Fisher</td>
			<td>4641170N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Micropipetter - 1000 uL&#160;</td>
			<td>Thermo Fisher</td>
			<td>4641210N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Micropipetter - 200 uL&#160;</td>
			<td>Thermo Fisher</td>
			<td>4641230N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Papain - 0.22 Filtered</td>
			<td>Worthington Biochemical</td>
			<td>LK003178</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Penicillin-Streptomycin (Pen Strep)</td>
			<td>Thermo Fisher</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Petri dishes -100mm disposable</td>
			<td>Fisher Scientific</td>
			<td>08-757-100D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Petri dishes -100mm glass</td>
			<td>VWR</td>
			<td>10754-788</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Petri dishes -35 mm&#160;</td>
			<td>Fisher Scientific</td>
			<td>08-757-100A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Phosphate buffer saline (PBS) (1X)</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Plasma Cleaning System</td>
			<td>Plasma Etch, Inc.</td>
			<td>PE-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Poly-D-lysine hydrobromide (PDL)</td>
			<td>Sigma Aldrich</td>
			<td>P6407-5MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Polyethylene conical (centrifuge) tube -15 mL</td>
			<td>Fisher Scientific</td>
			<td>05-527-90</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Polypropylene conical (centrifuge) tube -50 mL</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Procedure masks</td>
			<td>Imco</td>
			<td>1530-imc</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Pyruvate</td>
			<td>Sigma Aldrich</td>
			<td>P4562-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Scalpel blades</td>
			<td>World Precision Instruments</td>
			<td>&#160;500237-G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:420px;">Scissors - iris</td>
			<td>World Precision Instruments</td>
			<td>14111-G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Scissors - large</td>
			<td>World Precision Instruments</td>
			<td>14214</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Scissors - small surgical</td>
			<td>World Precision Instruments</td>
			<td>&#160;501733-G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Serological pipette - sterile, 1 mL&#160;</td>
			<td>VWR</td>
			<td>89130-892</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Serological pipette - sterile, 2 mL&#160;</td>
			<td>VWR</td>
			<td>89130-894</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Serological pipette - sterile, 25 mL&#160;</td>
			<td>VWR</td>
			<td>89130-900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Serological pipette - sterile, 50 mL&#160;</td>
			<td>VWR</td>
			<td>89130-902</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Software: Matlab GUI</td>
			<td>Peixoto Lab</td>
			<td>npeixoto@gmu.edu</td>
			<td>Used to analyze .mat files.&#160; Available for free upon request.&#160; Contact npeixoto@gmu.edu to request a copy.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Software: MCS MC_Rack</td>
			<td>MultiChannel System</td>
			<td>N/A</td>
			<td>Used for data acquisition</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Software: NeuroExplorer</td>
			<td>Plexon&#160;</td>
			<td>http://www.plexon.com/products/neuroexplorer</td>
			<td>Used to convert .nex files to .mat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Software: Offline Sorter</td>
			<td>Plexon&#160;</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 height="21">
			<td height="21" style="height:21px;width:420px;">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 height="21">
			<td height="21" style="height:21px;width:420px;">Software Manual: NeuroExplorer</td>
			<td>Plexon&#160;</td>
			<td></td>
			<td>https://www.neuroexplorer.com/downloads/Nex3Manual.pdf</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Software Manual: Offline Sorter</td>
			<td>Plexon&#160;</td>
			<td></td>
			<td>www.plexon.com/system/files/downloads/Offline%20Sorter%20v2.8%20Manual.pdf</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Spatula - small double-ended</td>
			<td>World Precision Instruments</td>
			<td>&#160;503440</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Stericup 0.22 &#181;m pore filter - 250 mL</td>
			<td>Millipore</td>
			<td>SCGVU02RE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Transfer pipette - large bore&#160;</td>
			<td>Thermo Fisher</td>
			<td>335-1S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Transfer pipette - small bore&#160;</td>
			<td>Thermo Fisher</td>
			<td>242-1S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">Trypan blue</td>
			<td>Sigma Aldrich</td>
			<td>T8154-20ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">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

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

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

结合经颅磁刺激和功能磁共振成像检查默认模式网络

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

科研

神经科学

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

NeuroStar TMS的设备进行美国FDA批准用于治疗抑郁症的议定书"

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

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 ...

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

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

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

谷氨酸和GABA快速微离子导入:一个有用​​的工具，调查突触整合

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

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

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

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

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

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

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

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

一个程序植入微丝的有组织阵列的单机录音清醒，举止动物

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

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 ...

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.

在斑马鱼幼虫的视觉反应的视网膜电图分析

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the ...

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 ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...