作者感谢露丝Rubli在瑞士伯尔尼大学的生理学系与实验宝贵的技术帮助。这项工作是由欧盟FP7-NMP程序（。 - www.nanoci.org项目NANOCI赠款协议没有281056）的部分资助。

动物护理和实验是根据瑞士当地政府（AMT献给Landwirtschaft UND NATUR德冠德伯尔尼，瑞士）的指导方针进行。 1.准备实验方案<ol><li>准备外基质（ECM）涂布液（见材料表，6点）:在冰上解冻ECM组合。稀释在基本培养基中的细胞外基质混合物1:10（无神经营养因子或胎牛血清（FBS）），并储存在冰上。 </li><li>准备培养基（见材料表 ，1点）:准备不含有FBS或脑源性神经营养因子（BDNF）的Neurobasal介质的股票。补充只添加到细胞培养物之前，新鲜的FBS（10％）和BDNF（5毫微克/毫升）的Neurobasal介质。 </li><li>准备细胞外液（见材料表 ，2点）。 </li></ol>多边环境协定2.清洗灭菌图1E）68电极。每个电极的大小为40×40微米2为200微米的从中心到中心的间隔。电极是由铂制成。的电极是通过由氧化铟锡的电路连接到相应的触点。此电路由SU-8的一个5微米的层绝缘。请参阅材料表上提供的详细信息。其他的MEA的布局可以是适合于这些实验。 <ol><li>为新的MEA:通过在70％乙醇中浸渍30秒冲洗它们，并用蒸馏水洗涤30秒。在层流罩的工作。 </li><li>让用于在层流罩30分钟在MEA干燥。 </li><li>把每个MEA到个体无菌培养皿（35毫米×10毫米），并用箔片封口。多边环境协定可存放待用。 </li><li>二手MEAS:孵育含有1ml酶SOLUT的培养皿（35毫米×10毫米）的多边环境离子（见材料表 ，6点）O / N上轨道摇床在室温除去生物材料。然后继续按步骤2.1。 注:用橡胶覆盖的提示，钳子小心处理多边环境协定。 </li></ol> 3.多边环境协定的文化实验的制备<ol><li>去除密封箔和离开MEA在培养皿（35毫米×10毫米），用于在整个实验。 </li><li>涂层具有在1.1中制备的溶液中的MEA:使用冷200μl的枪头（储存在-20℃）以吸取50微升涂布液到每个MEA中，覆盖整个电极面积。 </li><li>允许的MEA涂覆30分钟到1小时在室温。 </li><li>除去用吸管的涂布溶液。应用100微升培养基的补充有10％FBS和5NG / ml的BDNF和离开它在RT直至镀敷组织。 </li><li>将含有涂层多边环境协定成一个大的培养皿（94毫米×16毫米）2培养皿，并添加含1.5第三小培养皿毫升磷酸盐缓冲盐水（PBS）中加湿。 注意:添加含PBS的小皮氏培养皿（步骤3.5）是至关重要的显著最小化培养基的蒸发。 </li></ol> 4，螺旋神经节解剖注:总可以剥离层流罩外完成（步骤4.1到4.4）。对于精细解剖无菌条件下（层流罩）是强制性的（步骤4.5）。 <ol><li>动物实施安乐死（5-7日龄小鼠）断头，恕不另行麻醉。 </li><li>通过用70％乙醇喷雾消毒的头部。 </li><li>通过保持头，切开皮肤，并与沿矢状线尖锐/锐利剪刀颅骨之间的连接。 </li><li>矢状切头骨和使用锋利的/钝的剪刀取出大脑。 </li><li>从头骨切割颞骨并放置在那些含有无菌冰冷的Hanks&#39;平衡盐溶液（HBSS）的培养皿。 </li><li>使用dissec化显微镜，用细镊子解剖鼓泡和隔离内耳。 </li><li>除去耳蜗的骨，使用细镊子。 </li><li> （SV）一起用钳持螺旋神经节（SG）和SV的基部，并慢慢地从底座解开SV -to-顶点取下螺旋韧带和血管纹</li><li>隔离尔蒂（OC）的机关，SG和蜗轴（ 图1A - C） </li><li>通过用钳子夹持SG和所述OC的基部和慢慢展开从基站至顶点所述OC分开的SG和蜗轴所述OC。 </li><li>从螺旋神经节切横向外植体（直径为200〜500微米）仍然附连到使用镊子和微型手术刀蜗轴（ 图1D）。 </li></ol> 5.螺旋神经节外植体培养多边环境协定<ol><li>广场旁电极先前与10准备的MEA两个螺旋神经节外植体0微升培养基。 </li><li>放置Corti器从电极面积约5mm远。 </li><li>使用镊子的外植体和Corti器的针上的MEA，同时避免损坏组织。 </li><li>小心地将的MEA进入培养箱并培养在37℃和5％的CO 2。第二天，在视觉上检查该外植体已经附着到MEA。 注:如果植还没有连接的O / N，他们很少会做这样在接下来的日子里。业主立案法团被放在毗邻文化的营养/神经营养支持。 </li><li>加入100微升每日10％FBS和BDNF的含有连续5天的培养基。 </li><li>在第6天加入含10％FBS和5NG / ml的BDNF和文化组织额外13天培养液2毫升。 </li></ol> 6.电生理记录调查自发和电极刺激相关活动<ol><li>与细胞外洗MEA文化，使lution在步骤1.3制得，在RT。 </li><li>干用纸巾接触并安装在MEA的设置MEA。 注:要保持文化潮湿的安装过程中，加外解决了文化的一小滴。 </li><li>加入300微升细胞外溶液和记录之前等待10分钟，以允许系统稳定。 </li><li>从通过按下记录所有电极2分钟记录自发活动/获得该软件的按钮，并确定记录电极。 </li><li> MEA电极刺激:选择合适的软件刺激的幅度/时间/形状，适用于多种电极连续描述13。基础上选择的那些表示自发性活动的电极（如在步骤6.4）。从所有剩余的电极记录。 </li><li>要排除刺激伪迹，从同一个电极刺激的10倍。如果培养进行响应的至少8出10倍，它可以被假定为在电极刺激诱发的积极响应。 </li><li>以确定背景噪声，应用河豚毒素（TTX）到cultureat浓度1μM的，阻断电压门控钠通道和记录为2分钟。使用此执行尖峰检测（7.1和7.2）。 </li></ol> 7.数据分析注意:数据分析已经详细在图6和5如前所述。在这项研究中使用的软件的细节见材料表 ，7点。 <ol><li>使用合适的软件检测自发活动对于采用基于标准差和随后的鉴探测器每个电极。此过程在6进行说明。活动出现快速电压瞬变（&lt;5毫秒）。 </li><li>选择分析每个实验处理samples.Adapt的阈值时，TTX假positiv区分导致在没有活动的阈值e和假阴性检测。 </li><li>使用适当的软件按照标准方法（ - C见图2A）观察各电极的检测神经元活性为栅格图。 </li><li>确定并通过总结的10毫秒的滑动窗口内的所有检测到的事件，由1毫秒步骤错开显示总的网络活动。 </li><li>通过使用合适的分析软件显示的原始数据检测刺激诱发活性。离线手动识别单个尖峰。单尖峰出现快速电压瞬变，刺激神器（箭头图2E）后发生。一个例子示于图2E。 </li><li>根据实验，分析响应电极的数量，实现了响应所需要的阈值，每电极和感兴趣5其他参数动作电位的数目。 </li></ol> 8.组织固定和入境事务处unohistochemistry 注:染色工艺会破坏MEA。见试剂材料表 （4点）。 <ol><li>录音后直接清洗与文化37℃的温PBS三次。丢弃PBS中，并应用4％多聚甲醛（PFA）（在PBS中），预热至37℃10分钟。 注意:PFA是有毒的。在化学罩与根据指引适当的保护和丢弃的解决方案的工作。 </li><li>洗培养物三次，用PBS，并用2％牛血清白蛋白（BSA）的PBS（0.01％的Triton-X100）2小时阻塞。 </li><li>添加第一抗体（抗βIII微管蛋白，TUJ抗体）的PBS（2％BSA和0.01％的Triton-X100）稀释，并离开O / N在4℃。用PBS洗涤培养三次。 注意:从现在起保持样品在黑暗中尽可能经常以最小化二次荧光标记的抗体的漂白。 </li><li>添加二次抗体，在PBS中稀释（2％BSA的D 0.01％的Triton-X100），并孵育1至2小时在RT。染细胞核添加1:10,000的DAPI（1毫克/毫升的库存）10分钟。 </li><li>用PBS洗三次，向后安装样本，以薄盖玻片（24×50毫米2）使用安装介质（见材料表 ，4点）。图像使用带有5倍和20倍的放大倍率荧光显微镜。 </li></ol>

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The authors have nothing to disclose.

这里描述的协议显示了如何在文化MEA SGN外植体和细胞外无创记录评估SGN活动。这个平台，我们最近开发5允许对新刺激方案和电极材料从而减少能源需求的识别，激活SGN，为进一步实施人工耳蜗等神经修复术的潜在兴趣的协议。在介绍过程一些预防措施是准确的和可重复的实验成就的根本。 螺旋神经节和主要组织进一步的处理小心拨开需要特别谨慎。这些实验已经使用图5和7天前之间的C57 / BL6小鼠中进行。在相同的年龄范围使用Wistar大鼠获得类似的结果（未显示数据）。我们相信这是精细解剖的最佳年龄为耳蜗骨仍是软连接ough容易去除由镊子，和科尔蒂的螺旋神经节和器官可以在不破裂树枝状进程或SGN胞体容易地分离。在较早的年龄的组织过于柔软，机会翻录SGN胞体连同柯蒂氏器是高，而在后期阶段，耳蜗骨胶囊的硬化增加损伤组织而解剖的危险。对SG的适当隔离以及外植体的仔细切割是至关重要的。这些应在范围200〜500微米的大小，以附着最大化到MEA表面，并有在所述外植体，从该突起将再生足够数目的SG胞体的。以增加神经营养支持在培养的最初几天，新鲜的BDNF每天加入柯蒂氏器被放置在共培养。 夹层的速度也很重要。所有步骤应以最小化组织劣化迅速，使用冰冷的溶液来进行。该安乐死和放置外植体在MEA之间的时间应该是10-15分钟之间，并且是成功的培养物是至关重要的。让所有的工具准备好，以避免在文化镀延误。 所有步骤，包括精细解剖，将培养的维护和制备介质和设备都是在无菌条件下进行的。当与ECM溶液涂布在MEA解冻它在冰上并在较高温度下使用冰冷枪头和冰冷介质作为ECM的混合物的凝胶是非常重要的。当将植上的多边环境协定，第一介质添加到电极区域，随后放置外植体。如果介质在第二步骤中加入，外植体趋于分离从MEA由于剪切应力。因为介质的小体积在第5天施加到培养，培养基的蒸发，必须最小化。因此，强烈建议创建使用含PBS密切Proxim的小培养皿加湿室性的多边环境协定。 这里所描述的实验，利用市售的MEA的电极与特定的电极尺寸，涂层材料和帧内电极的距离（如在点2所述，注）进行。培养条件在这里已经进行了优化，以最大化特定电极网格设计的神经元的覆盖。这是可能的的电极间距，几何形状和表面其它配置可能需要不同的涂层或细胞密度。这些步骤可能要求初始故障处理，以达到高密度培养物。 关于电生理记录，在开始录像的培养是从培养基中在37℃下在RT转移到细胞外溶液中之前。为了避免不稳定的录音，等待约10分钟，使文化保持稳定。当刺激的文化，特别应谨慎在选择刺激使用作为大振幅（&gt; 3 V）和持续时间可能会导致在培养的损坏。如对刺激脉冲的形状和持续时间的参考见参考文献5。 用于数据采集和处理自制硬件（记录室中，到放大器和放大器连接）被用来与被选择的特定分析程序（见材料表 ，点7）。然而，其他商业MEA设置和其他软件包适合以及对这些操作。之前我们已经评估我们的文化的答复在3个不同的时间点，且随时间延长培养时间5活性增加。在这里，我们建议18天体外录音。 MEA系统允许连续记录在无菌，加湿室，可以促进对每个培养类型的最佳的时间点的识别。 这种生物测定的主要缺点是在T高变异他数的每培养电极，显示对刺激的反应。的刺激响应这个速率主要取决于四个因素:在培养物中的神经突的密度，突起和电极，所述突起或突起束的直径，并且电极的阻抗之间的联系。关于突起的密度，只有生长在至少两个电极突起可以用于刺激实验。突起和电极之间的接触依赖于周围组织，可以一方面隔离电极的突起，因而恶化了接触，或者，另一方面，隔离突起和从周围浴的电极，从而改善接触。组织密度，以及突起的大小，由所使用的外植体和培养条件限定。解离的神经元培养物也已成功地进行测试，但在这些培养的​​神经元密度低得多，从而导致RECO的数量减少录制电极。因此，细胞培养物应适应要处理的具体的科学问题。 最后，该电极的阻抗主要由大小和电极的表面上给出。材料，形成一个大的表面，如铂黑， 如图3，降低了电极的阻抗也可提高电极和突起7-9之间的耦合。 因此策略提高刺激的成功率包括的培养条件的优化，总电极或电极密度的数量的增加与电极表面10的调制。 截至目前，已经利用膜片钳技术进行SG神经元的电生理特性。这使得动作电位的细胞内记录和细胞内的离子电流的详细分析单个神经元。这里，我们提出的体外生物测定可用于通过同时分析自发活动或响应许多神经元的细胞外的刺激来研究螺旋神经节神经元的活性谱。此外，电极和螺旋神经节神经元之间的相互作用，可以研究和修改的或新的材料的应用进行优化。最后，即使这里没有示出，该平台可组合使用的外部电极，安装在显微最近由我们的组5所示，在为了研究刺激电极和文化活动的距离之间的关系。所有这些新颖的方面允许的耳蜗植入电极的主要功能的模仿可能导致新的假体装置的设计。 我们的模型是一种在体外工具非常有用的研究策略，提高听神经元，并进一步运算的刺激功效timize CI技术。一旦技术被掌握，人们可以设想筛选一些变量的修改:一个）的不同神经元群体，二）不同电极材料/尺寸/阻抗三）执行慢性实验来检验材料毒性或电极刺激诱导的毒性，这可以对体内电极阵列更安全，更有效的刺激方案线索。

In accordance with the World Health Organization, 360 million people worldwide suffer from hearing loss with profound consequences on professional and private life. Hearing aids can restore sensory function in moderate forms of hearing loss; however, for the most severe cases, the most effective treatment option is a prosthetic device called a cochlear implant (CI). CIs contain a linear electrode array of up to 24 electrodes, which is surgically inserted into the scala tympani of the cochlea. The electrodes directly stimulate the spiral ganglion neurons, forming the auditory nerve 1. 
With more than 300,000 devices implanted worldwide, CIs are very successful medical implants and rank among the most cost-effective procedure ever reported. Despite its success the cochlear implant still has limitations such as reduced frequency resolution compared to physiological hearing. This can lead to deficits in effective communication in groups or noisy environments, and the ability to decipher very complex sounds such as music. This reduced frequency resolution is likely due to the gap between the CI electrodes and the spiral ganglion neurons, leading to stimulation of large groups of neurons. This gap is in the range of hundreds of micrometers 2,3. Elimination of this gap would facilitate the stimulation of smaller groups of neurons per electrode, thereby increasing frequency resolution and overall performance of the device 4.
To study the influence of the gap between the electrode and the neuron and the effect of various optimized stimulation protocols, we have developed an in vitro bioassay based on a non-invasive electrophysiological characterization of SGNs on multi electrode arrays (MEAs) 5. Additionally, MEAs can be easily modified to vary electrode shape, size, material and surface roughness, to optimize the neuron-electrode interface. The following is a step-by-step protocol to reproducibly obtain recordings from murine spiral ganglion neuron cultures and assess the dependency on the above-mentioned parameters.

<table><tbody><tr><td>&lt;strong&gt;culture medium&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Neurobasal medium</td><td>Invitrogen</td><td>21103-049</td><td>24 ml (for 25 ml)</td></tr><tr><td>HEPES</td><td>Invitrogen</td><td>15630-080</td><td>250 &amp;mu;l (for 25 ml)</td></tr><tr><td>Glutamax</td><td>Invitrogen</td><td>35050-061</td><td>250 &amp;mu;l (for 25 ml)</td></tr><tr><td>B27</td><td>Invitrogen</td><td>17504-044</td><td>500 &amp;mu;l (for 25 ml)</td></tr><tr><td>FBS</td><td>GIBCO</td><td>10099-141</td><td>10% (for 25 ml)</td></tr><tr><td>BDNF</td><td>R&amp;amp;D Systems</td><td>248-BD-025/CF</td><td>final 5 ng/ml (for 25 ml)</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;extracellular solution (pH 7.4)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NaCl</td><td></td><td></td><td>145 mM</td></tr><tr><td>KCl</td><td></td><td></td><td>4 mM</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>1 mM</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td>2 mM</td></tr><tr><td>HEPES</td><td></td><td></td><td>5 mM</td></tr><tr><td>Na-pyruvate</td><td></td><td></td><td>2 mM</td></tr><tr><td>Glucose</td><td></td><td></td><td>5 mM</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;blocking solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PBS</td><td>Invitrogen</td><td>10010023</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A4503-50G</td><td>2%</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100</td><td>0.01%</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Immunostaining solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>TuJ</td><td>R&amp;amp;D Systems</td><td>MAB1195</td><td>dil 1:200</td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9542</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>158127</td><td>4%</td></tr><tr><td>Fluoreshild</td><td>Sigma</td><td>F6057</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;plastic/tools&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Petri dish 35 mm</td><td>Huberlab</td><td>7.627 102</td><td></td></tr><tr><td>Petri dish 94 mm</td><td>Huberlab</td><td>7.633 180</td><td></td></tr><tr><td>Dumont #5 tweezer</td><td>WPI</td><td>14098</td><td></td></tr><tr><td>Dumont #55 tweezer</td><td>WPI</td><td>14099</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Materials&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Enzymatic solution: Terg-a-Zyme</td><td>Sigma</td><td>Z273287-11KG</td><td></td></tr><tr><td>Extracellular Matrix (ECM) mix: Matrigel TM</td><td>Corning</td><td>356230</td><td></td></tr><tr><td>MEA electrodes</td><td>Qwane Biosciences</td><td></td><td>(Lausanne, Switzerland)</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Labview</td><td>National Instruments</td><td></td><td>Switzerland</td></tr><tr><td>IgorPro</td><td>WaveMetrics</td><td></td><td>Lake Oswega, USA</td></tr></tbody></table>

spiral ganglion neuron explant culture and electrophysiology on multi electrode arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. 
We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

图1总结了组织分离，制备和文化上的MEA的过程。我们表明组织剥离的连续步骤以分离从科尔蒂（OC）和血管纹（SV）的器官感觉上皮螺旋神经节（SG）和所附螺旋韧带（ 图1的 - C）。螺旋神经节外植体（3-4号）切断与微型剪刀从神经节， 如图1D示意性示出，并放置在MEA（ 图1E），在电极所占的面积（2.2 平方毫米）。所述OC被放置在接近时，电极表面的外侧。培养物的生长可在一段时间（ 图1G）进行监测。该协议的示意图见图1楼 。电生理学活性可以后6天培养的，其与延长培养时间增加来检测。我们recommend评估18天文化即产生显著更高的数字记录电极相比，较早的时间点5。 <img alt="图1" src="/files/ftp_upload/54538/54538fig1.jpg" /> 图1.细胞培养的准备。（A）新鲜解剖小鼠内耳。白色虚线表示耳蜗的位置，黑虚线表示前庭区域。除去耳蜗骨壁的后（B）的小鼠内耳。耳蜗匝由白虚线表示。 （C）螺旋神经节（SG）和蜗轴，尔蒂（OC）和血管纹（SV）和螺旋韧带的器官的解剖后显示。 （D）SG和OC解剖和SG外植体的制备{图改编自5经批准从出版商}示意图。在此研究中使用的多电极阵列（E）的示意图。重新编码电极在中心矩形网格组织占据为2.2mm 2的面积。 4接地电极和侧面接触所示。文化协议（F）示意图。录音是在18天（G）代表的图片（亮场图像），并在第1天，第6和18比例尺= 400微米文化监视MEA SG外植体方案执行。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54538/54538fig1large.jpg" target="_blank">请点击此处查看大图这个数字。</a> 自发活动可以在MEA进行检测和显示为栅格图，其中的情节的每一行代表一个检测秒杀。示（在图不同的颜色）从几个电极检测自发性活动的代表性示例示出（ 图2A，2B和2C）。 图2D）的双相脉冲的代表性示例，5响应电极（红色迹线）和无应答的电极（蓝色迹线）示于图2E。对于这些实验被用于刺激和所有其它电极用于记录一个电极。单动作电位的刺激伪迹（黑色箭头）后，出现了1毫秒。在MEA可以在该过程的结束，以评估在电极区域中的神经元突起的覆盖进行免疫染色。用于刺激在绿色在图2F所示的电极，并以红色指示的电极用于记录响应（ 图2E）。 <img alt="图2" src="/files/ftp_upload/54538/54538fig2.jpg" /> 图2. MEA数据记录。（A &lt;/ STRONG&gt;）六出于63电极呈现自发活动的原始记录的痕迹。尖峰检测之后图2A的六个电极的（B）的光栅图。每个条形代表一个动作电位。包括所有电极（如A和B）活动（c）光栅地块从63电极（通道数0-63）2分入账。 （D）一种双相刺激具有80微秒的总持续时间为80微安振幅被用于从一个电极（E58 图2F）培养的刺激。 （E）从电极58示出的动作电位（红色迹线）刺激后获得的原始数据曲线的代表性例子或无刺激后的反应（蓝色迹线）（黑色箭头头部）。 （F）在MEA免疫染色的神经元标记TUJ（绿色）螺旋神经节文化在实验形象化神经月底<html>电极面积的最终覆盖{图改编自5与出版商批准}。用于刺激电极58以绿色指示，响应电极用红色表示。比例尺= 50微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54538/54538fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 最后，MEA设置允许为研究可能的电极表面改性可以增加记录灵敏度。两种市售的MEA电极在该研究中使用两种不同的铂表面:灰色铂（灰铂），其中包括一个150纳米厚的Pt层（阻抗:400KΩ/ 1千赫）的和黑色铂（黑，铂），由铂的电化学沉积在微制造工艺（20KΩ/ 1千赫阻抗）结束时获得的。详情请参阅材料表 （6点）。 ENT"FO:保持-together.within页="1"&gt;每极型进行6个独立的MEA实验黑色铂MEA允许在一个更高的数目的每MEA（ 图3A）的电极检测神经元活性和减少的。引出一个响应（ 图3B）所需要的激励振幅，我们分析了30-35独立电极对，在6个不同的实验中，以实现响应需要的电流阈值。黑色的Pt的电极进行显著更好示出31.09μA的阈值±2.4相比，灰色铂电极47.57μA +/- 1.97。 <img alt="图3" src="/files/ftp_upload/54538/54538fig3.jpg" /> 图VS黑色铂电极灰色3.比较两个类型的电极（灰色和黑色的Pt）进行比较，通过使用12个独立的MEA文化，6各MEA类型相映成趣。 （A）重的总数每个实验应的电极被示出。 （B）的振幅的阈值，以引起反应，使用在6个独立的MEA实验30-35独立电极对测量被示出。数据显示为平均值+/- SD。 （学生t检验P &lt;0.001）， <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54538/54538fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays at JoVE.com

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

螺旋神经节神经元外植体培养及电多电极阵列

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...

spiral-ganglion-neuron-explant-culture-electrophysiology-on-multi

Research

JoVE Journal

Neuroscience

9.8K 次观看.  Inselspital and University of Bern. We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

视频: Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi 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

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6.
Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. 
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

小鸡Statoacoustic神经节和脊髓植在胶原凝胶夹层和文化突起生长实验

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.

Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.

全细胞膜片钳红外神经刺激机制的调查

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

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 ...

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.

Presented here are methods providing detailed instructions for dissecting, dissociating, culturing, and patch-clamp recording from vestibular ganglion and spiral ganglion neurons of the inner ear.

从新生儿啮齿动物中分离和培养前庭和螺旋神经节 Somata 以进行膜片钳记录

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter ...

Analyzing Neural Activity in Primary Murine Spiral Ganglion Neurons Using Multielectrode Arrays

Source: Hahnewald, S., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54538">Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays.</a> J. Vis. Exp. (2016).
This video demonstrates the recording of spontaneous and stimulation-dependent neural activity from primary murine spiral ganglion neuron (SGN) cultures on a multielectrode array (MEA). It involves washing and preparing the MEA, recording spontaneous neural activity, and applying controlled electrical stimuli to identify stimulation-dependent neuronal activity.

使用多电极阵列分析原代小鼠螺旋神经节神经元中的神经活动

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Electrophysiological ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Culturing Murine Spiral Ganglion Neuron Explants on Multielectrode Arrays

Source: Hahnewald, S., et al <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54538">Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays.</a> J. Vis. Exp. (2016)
This video demonstrates the process for isolating spiral ganglion explants from the inner ear and co-culturing them with the organ of Corti on a multielectrode array. Spiral ganglia are carefully extracted and sectioned into smaller tissue sections or explants. These explants are cultured on the multielectrode array&#39;s active area to promote neuronal process outgrowth.

在多电极阵列上培养小鼠螺旋神经节神经元外植体

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Prepare Solutions for 
...

Neuronal Culture Techniques

Primary Neuronal Culture

Isolating and Culturing Chick Embryonic Neurons on a Multi-Electrode Array

Source: Sanchez, K. R., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56300">Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays.</a> J. Vis. Exp.(2018)
This video demonstrates a method to isolate neurons from the forebrain of an early-stage chick embryo and culture them on a microelectrode array to form a neuronal network.

在多电极阵列上分离和培养雏鸡胚胎神经元

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Advanced Neuronal Models

Electrophysiological Recordings For Assessing Neuronal Regenerations in Co-cultured Spinal Cord Slices

Put a Petri dish with a coated microelectrode array inside under a stereomicroscope. Bring the array into focus, and center a six-microliter droplet of chicken plasma on the electrode array. Using a small spatula, carefully slide two spinal cord sections with their ventral sides facing each other into the plasma droplet.
Next, add eight microliters of thrombin around the chicken plasma droplet. Then, use the pipette tip to carefully mix and spread the chicken plasma and thrombin mixture. Just before the chicken plasma and thrombin mixture becomes too stringy and starts to coagulate, aspirate the excess liquid. Then, cap the Petri dish and place it in a humidified chamber.
Place the chamber inside an incubator at 37 degrees Celsius for about an hour. After incubation, carefully add 10 microliters of nutrient medium to the sample. Cap the Petri dish and place it back into the incubator for an additional 45 minutes. Next, place each of the multi-electrode array culture assemblies into a roller tube and add three milliliters of nutrient medium. Close the lid tightly and place the roller tube into the roller drum. Rotate the drum at 1 to 2 RPM in the incubator at 37 degrees Celsius.
Using sterile rubber-tipped forceps, remove the multi-electrode array culture assemblies from the roller tube and place them into a Petri dish under a stereomicroscope. Bring the tissue into focus and verify that the two slices are fused. Next, hold the assembly steady and place a scalpel blade in the groove of the multielectrode array close to the tissue slices. Hold the scalpel rather horizontally and then lift the scalpel handle up, but let the scalpel blade stay in the groove of the array in such a way that the blade rolls from base to tip, cutting through the tissue, covering the groove.
Sever any residual tissue connections with a 25-gauge needle tip if necessary. Work only in the area within the groove, and do not touch the tender edges. Put the multi-electrode array culture assemblies back into the roller tube and add three milliliters of fresh nutrient medium to the cultures. Then, place the roller tube back onto the roller drum in the incubator at 37 degrees Celsius.
Mount a multi-electrode array culture assembly into a recording chamber and apply about 500 microliters of extracellular solution to the array. Then, mount the assembly on the microscope. Wait 10 minutes for the system to stabilize, and then, record basic spontaneous activity from each of the activity-detecting electrodes for 10 minutes. Repeat the recordings a total of two times.
To ensure stable extracellular conditions, exchange the extracellular solution after every recording session. If desired, disinhibit the network by applying extracellular solution containing one micromolar of strychnine and 10 micromolar of gabazine and wait for at least two minutes before recording the electrical activity.

用于评估共培养脊髓切片中神经元再生的电生理记录

EoE Sub Articles

Utilizing Multielectrode Arrays to Measure Synaptic Physiology in 3D Coculture Spheres

Begin by coating the surface of each multielectrode array or MEA with 1 millimeter of poly-ornithine to render the surface hydrophilic and incubate at 37 degrees Celsius for a minimum of four hours. Following the incubation, remove the poly-ornithine and wash the surface of each MEA with deionized water. Then, add 1 milliliter of extracellular matrix and return to the incubator for a minimum of four hours.
When ready, remove the extracellular matrix, then apply the spheres in 1.5 milliliters of medium onto the MEA surface, ensuring that the spheres are positioned on top of the electrode array. Allow to adhere for two days. To measure the electrical activity of neural spheres, place the MEA on a temperature-controlled head stage.