由NINDS 5P30NS069250支持这项工作。我们感谢大雅纳Chehayeb，杰弗里·辛普森，泰勒罗森鲍姆和克里斯托弗·胡佛的技术援助。

实施耳机系统由四个主要步骤。第1节下面的信息耳机帧的组装，该收纳的电子设备（扬声器和一个小型化的麦克风）。第2节描述帧是如何连接到鸟。第3节介绍了组装的电子产品。第4节说明如何将电子设备连接到声音处理和数据采集硬件和细节测试，该系统运行正常的程序。 1。制造耳机框架<ol><li>切碳纤维组件的横杆和耳机。使用切割轮上Dremel工具准备以下几部分: 1横杆（3厘米×3毫米的碳纤维条） 2个垂直支柱（1.5厘米×3毫米的碳纤维条） 2耳塞（4毫米，4毫米的ID / 6 mm外径碳纤维气瓶） </li><li>组装横梁。钻两个小孔的4.5毫米除了在中心的横杆（ <s仲&gt;图1a中，顶部）使用1.4毫米直径的位。孔应足够大，以容纳0-80螺丝。虽然孔，并附上插入两个0-80螺钉，六角螺母。切两毫米的条注射器针头（任何小直径的针，将工作），并压平，用钳子（针带红色的图1a）。将横梁过来，让躺在上面的六角螺母。为了防止环氧树脂从胶合的六角螺母的螺丝的前端把一滴矿物油（绿色在图1a中所示），每根螺杆的前端上。将两针段的顶部的六角螺母。彼此的环氧六角螺母和针段，涵盖了（ 图1a，底部）。环氧树脂完全固化后（24小时），删除这两个螺丝。横杆单元应该分成四部分组成:两个螺钉，3厘米长的碳纤维，和一个单件由用环氧树脂粘合在一起的2个六角螺母和2针分部。重新安装横梁单元。&lt;/ P&gt; <li>制造泡沫环。使用1/4英寸（6.35毫米）打孔切割泡沫筒从泡沫耳塞。使用剪刀剪下得到2毫米（长度）由6.35毫米（直径）气缸的泡沫滚筒的长轴垂直。然后，使用1/8英寸（3.175毫米）打孔机切出2毫米泡沫缸的中心，形成一个的泡沫圈（以橙色显示在图1b）。 </li><li>组装耳塞。使用环氧树脂胶垂直支杆（1.5厘米长的碳纤维条带）具有4mm的长度，如在图1b中所示的碳纤维气瓶，底部。当环氧树脂硬化时，使用环氧树脂胶少量泡沫环的气缸的端部（ 图1b中 ，底部）。泡沫环鸟的头靠在。 </li></ol> 2。连接耳机帧鸟<ol><li>准备stereotax。在手术前，通过耳塞插入stereotax earbars，泡沫垫向内（ 图1c </strONG&gt;，上图）。 </li><li>连接横梁。使用异氟醚和/或氯胺酮咪达唑仑麻醉的鸟。麻醉鸟应该闭着眼睛，反应迟钝脚趾掐。将鸟stereotax，并有5-10毫米的切口在头皮。切口应沿中线延长前从一个点2毫米前的颈部肌肉附着在头骨的背面。用70％乙醇干燥骨表面擦暴露颅骨。使用弹簧夹支架放置横杆等环氧覆盖的六角螺母其余的头骨的顶部上（ 图1c，四 ）。提起横梁稍稍胶六角螺母（与环氧树脂或牙科丙烯酸）的头骨，要小心这样的粘接接触六角螺母，而不是横梁本身。这将允许您取下耳机，只留下永久固定头（ 图1d）的螺母拆下螺丝。 </li><li>连接耳机。幻灯片耳塞ALON克耳棒，直到泡沫垫对鸟的头部（ 图1c，上图）。使用鳄鱼夹在耳朵上条按泡沫垫，坚决反对鸟的头部（ 图1c，下图），这是很重要的，以获得一个良好的声学密封。旋转耳塞的垂直职位碰横梁的背面，留出空间后前的接线板插座（ 图2）。使用环氧树脂或丙烯酸胶职位，以交叉开关。 </li><li>恢复。允许24小时，完全固化的环氧树脂/丙烯酸和鸟类恢复。恢复后，检查，以确保泡沫垫耳道周围形成一个密封的框架。如果密封是松散的，泡沫垫，可以删除和替换较大的提高防声封条。轻轻地使用0.05英寸（1.27毫米）六角扳手卸下螺丝和删除组装耳机帧的鸟。 </li></ol> 3。装配电子产品<ol><li>制造音箱适配器。使扬声器适配器通过插入枪头（5.2厘米长的前端与从5毫米到1毫米的直径，其逐渐变细）成废料片的碳纤维气瓶（使用相同的4 mm内径/ 6毫米外径cyinder库存为耳塞;长度气缸废料是并不重要），然后切成枪头1毫米的气缸（ 图2a）的过去的结束。移除缸和切割，从而使适配器的总长度为3毫米。 </li><li>连接扬声器。焊接5厘米长的绝缘36 GA。两个扬声器（绿线和黑线，分别在图2d中，电子 ）的正极和负极端子的导线。用欧姆表验证既不终端是在扬声器的金属外壳的电气接触。 </li><li>装配扬声器/麦克风组成部分。环氧树脂的磁带的带状（任何电绝缘带将工作）的一个侧的一个扬声器和环氧树脂的顶部的带（ 图2b）的耳机麦克风。焊接地线来自麦克风的负面吨erminal上的扬声器（连接在图2e中所示的黑色）。 </li><li>环氧两个扬声器和扬声器/麦克风组件到每个适配器的广角端（ 图2c，右）。的扬声器和麦克风端口（只）放入吸管。确保环氧树脂不包括任何一个端口。 </li><li>轻轻地插入适配器插入耳塞（ 图2d，右）。每个适配器连接到它的耳塞胶滴一小滴环氧树脂。 </li><li>制造接线板。切段组成的四个端子的连接器条插座和放置一个参考标记（ 图2d中的白点）的插槽中的一个角部上。的垂直职位的接线板附近的位置。的扬声器和麦克风导线焊接在图2e中所示的配置中的销。用欧姆表检查接线板短路和修复，如果必要的。环氧树脂的连接器带耳机fRAME。周围裸露的电线，以保护电线和电气或透明胶带裹条添加薄薄的一层环氧树脂磁带两端，以防止解开。成品重量应为1.3-1.5克的耳机（包括帧和电子）。 </li><li>建立一个灵活的铅焊接的4个15厘米长的绝缘36 GA。导线连接器条标题的段组成的四个端子（ 图3a）。将一个参考标记（ 图3中的白点）的头，以确保在正确的方向插入插座的引线插入一个角落。编织物的线和通过焊接导线插入换向器（ 图3b）的一个适配器的光洁度铅。 </li></ol> 4。耳机电子产品连接到电源和信号处理设备<ol><li>收集，过滤，音调变化，以及，放大声音输入。直接一个挂一个全向电容式麦克风（"笼式麦克风， 图4）波夫鸟的笼子。 Preamplify和低通​​滤波该信号（截止在雀歌曲的10千赫），并将其输入到泛音（或其他实时声音处理硬件）。使用的音调移位器模块上的泛音产生一个移位的声学信号。放大该信号和路线（通过的换向器和灵活的铅）的正通道上的扬声器的耳机（绿色在图2e）。 </li><li>手机话筒提供电源。一节9V电池安装在墙上的鸟的共鸣。通过换向器和灵活的铅，电池的负极端子连接到一个共同的地面上的信号处理设备，该电池的正极端子连接到V +通道（红色在图2e）。将这个共同点，手机扬声器和麦克风地面（ 图2e黑色）。 </li><li>反馈增益校准。将成品耳机鸟的头部用0-80螺钉六角螺母。当鸟儿开始歌唱，RECORD三个通道的数据（笼麦克风/非移动信号，转向信号和电话麦克风）， 如图4所示。检查从手机麦克风记录的声音。该通道将记录叠加的虚拟反馈（通过耳机扬声器播放）和鸟的实际歌曲"泄漏"直接插入耳机框架。调整的幅度设置的扬声器放大器（ 图4），因此，虚拟听觉反馈是15-20分贝的声音比直接/泄漏信号。请注意，增加超过此临界值的振幅可以通过耳机和被拾起的轿厢麦克风，导致反馈失真导致的虚拟信号出血。良好校准的系统的一个例子（在其中记录的信号由耳机麦克风的间距偏移的信号，而不是直接/泄漏信号为主）在图5中示出。在这里，记录的信号通过耳机麦克风（ 图5a<html> ，右），主要是由的间距偏移的信号（而不是鸟的自然发声）所示，在图5b的例子中功率谱。 </li><li>监视系统的性能。检查耳机，每日两次，以确保扬声器和麦克风正常工作。需要注意的是耳机麦克风只能监视一个扬声器。要测试其他的扬声器，挖掘笼的麦克风而持有的非监控的扬声器，你的耳朵旁边 - 如果非监控扬声器工作，你会听到喀哒声。另一个建议的检查是手机的话筒，并使用欧姆表测量扬声器（黑色和绿色的图2e）之间的电阻断开电源和信号输出。两个扬声器被连接时，这将是自扬声器并联连接在单个扬声器之间的电阻的一半。 </li></ol> 5。材料成本的一个注记NT"有两个显着的例外，材料表中列出的所有项目的价格相对低廉（小于几百亿美元）。最昂贵的组件是换向器和表中列出的泛音，每个成本2000美元以上。我们注意到，更便宜的版本，这两个项目可以从不同的厂商比列出的那些（虽然我们还没有测试），可能使研究人员能够以较低的成本实现此协议。

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没有利益冲突的声明。

这里介绍的协议，允许实验者操纵听觉反馈的鸟鸣。轻巧的结构允许这样的操作将持续多长时间，并鸟儿会唱力强，同时为一个月或更长时间戴着耳机。虽然一些鸣禽唱戴着耳机，在某些情况下，只要10个星期的歌声开始下降，后5个星期的使用量。出于这个原因，我们通常会限制到4周的实验。根据我们的经验，每一个女歌手可以唱100 +首歌，每天发作（有时）配有耳机。因此，如果妥善运用，耳机系统提供了近100％的成功率（如果成功的定义是收购鸟鸣的数据）。此外，完成一个学习实验后，耳机可以被删除，随后进一步收集数据重新连接。在好一般健康reatta的动物是<html> chment可以发生在任何时间。 成功的重要的决定因素之一，是最小化的重量，和优化的耳机的舒适。在施工过程中，应注意尽量减少环氧树脂或牙科丙烯酸作为多余的粘合剂，将提高整个设备的重量，并有可能减少鸟唱的意愿。此外，连接耳机后数天，应简单地删除该设备，以验证耳道周围的皮肤变得恼火的耳塞，这可能发生，如果耳塞太紧张。耳道应该会出现，就像他们在的时候，耳机附件（开放，没有发红或肿胀的迹象）。若发生刺激，能够缓和压力通过减少泡沫垫的厚度。照顾到确保干环氧树脂硬化，泡沫不接触鸟类的皮肤，因为这也将造成刺激。 <html>吨"&gt;它是重要的以注意，除了的节距移位由实验者选择虚拟听觉反馈也延迟（〜10 ms的，反映的处理延迟的泛音）和在一个更大的振幅比鸟的介绍了自然的听觉反馈（为了以淹没了鸟类的天然歌曲"泄漏"插入耳机的声音）。出于这个原因，实验应该开始一个基线期的几天中，鸟唱与耳机上，但与零移调14日 ，允许在球场转移到被隔绝声音的变化等因素有关的耳机范例的影响，在实践中，在歌曲的音调或幅度的变化时，很少观察到的鸟第一次使用耳机的情况下开始唱的音调变化。此外，我们已经表明，长期暴露在非移动通过耳机传递的反馈意见不会导致间距14首歌曲的变化。 内容"我们以前使用过这种设计表明，在成年鸣禽，在球场上的听觉反馈向上和向下的变化产生适应性改变音高（ 即改变符号相反反馈移位）14。包括向上和采用这种模式的任何实验的向下转变是很重要的，因为这样的设计，可以显示出歌曲音高变化响应听觉反馈（而不是在响应于耳机引入的延迟或振幅工件）的间距中的变化，此外，一个密钥强度，这种模式是，它可以被用于，引入任意听觉操纵。泛音系统可以产生各种各样的在线扰动，例如，通过改变的振幅或音响信号的频谱包络线的扩大超出间距的范围内操作变化，可能因此被用于检查各种声乐学习的现象。此外，HEAdphones可以用来提供白噪声或其他条件加固信号来驱动学习在个别音节16。最后，这种模式可以在原则上可以采用任何小动物依赖于听觉反馈系统，在声音的行为。 我们注意到，我们的技术，用来研究人类的语言4-7模仿听觉反馈的操作，让声音的可塑性可查的生理访问的动物模型。结合可用于脑病变，药物处理，或神经录音的声音纠错行为研究揭示了特定的神经回路的声乐表演中的错误修正。

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>六角螺母</td><td>亚马逊供应</td><td> B000FMW43Y </td><td></td></tr><tr><td> 0-80螺丝，1/8" </td><td>亚马逊供应</td><td> B000FN0JXK </td><td></td></tr><tr><td> 0.05"六角扳手</td><td>亚马逊供应</td><td> B003GDISE8 </td><td></td></tr><tr><td>耳机喇叭</td><td> DIGIKEY </td><td> 423-1113-ND </td><td></td></tr><tr><td>耳机麦克风</td><td> DIGIKEY </td><td> 423-1062-ND </td><td></td></tr><tr><td>泛音</td><td>斯威特沃特声音</td><td> H7600</td><td></td></tr><tr><td> （黄昏H7600） </td><td></td><td></td><td></td></tr><tr><td>碳纤维带，1 x 3毫米; </td><td>国际业余爱好大堂</td><td> GXS1030 </td><td></td></tr><tr><td>碳纤维缸，6毫米（OD）x 4毫米（ID） </td><td>国际业余爱好大堂</td><td> GXT6040 </td><td></td></tr><tr><td>线</td><td> Cooner电线电缆</td><td> NUF36-2550 </td><td></td></tr><tr><td>接线板头</td><td> DIGIKEY </td><td> ED83100-ND </td><td></td></tr><tr><td>接线板插座</td><td> DIGIKEY </td><td> ED85100-ND </td><td></td></tr><tr><td>泡沫耳塞</td><td> AO安全</td><td> 92050 </td><td></td></tr><tr> &lt;TD&gt; 1/8"打孔</td><td> Paperwishes </td><td> 7260197000 </td><td></td></tr><tr><td> 1/4"打孔</td><td> Paperwishes </td><td> 7260198000 </td><td></td></tr><tr><td>枪头</td><td> VWR </td><td> 89003-056 </td><td></td></tr><tr><td>牙科丙烯酸</td><td> Maxcem </td><td> 33873 </td><td></td></tr><tr><td> 5分钟环氧</td><td> DEVCON </td><td> 14210 </td><td></td></tr><tr><td>笼式麦克风</td><td>乡下人</td><td> B3P4FF05B </td><td></td></tr><tr><td>麦克风前置放大器</td><td> M-AUDIO </td><td> DMP3 </td><td></td></tr><tr><td>扬声器放大器（皇冠D-45） </td><td>甜水的声音</td><td> D-45 </td><td></td></tr><tr><td>低通滤波器</td><td>克罗恩-HITE </td><td> FMB3002AC，3FS8SL-10KG N1U1 </td><td></td></tr><tr><td>换向器</td><td>蜻蜓</td><td> SL-88-10 </td><td></td></tr><tr><td>鳄鱼夹夹</td><td> GC电子产品</td><td> 12-051 </td><td></td></tr><tr><td>矿物油</td><td>西格玛</td><td> M3516 </td><td></td></tr><tr><td> Dremel工具</td><td> DREMEL </td><td> 8200 </td><td></td></tr></tbody></table>

a lightweight, headphones-based system for manipulating auditory feedback in songbirds

电机控制和感觉可塑性相关的计算，实验操作的感觉反馈，在复杂的行为提供了有价值的见解。一致的感官扰动导致代偿性改变电机的输出，反映了前馈电机控制，减少经验丰富的反馈错误。通过量化不同的感官反馈误差是如何影响人的行为，以前的研究已经探索了如何使用视觉信号校准手臂 ​​的动作2,3和听觉反馈将被用来修改言语产生4-7。这种方法的优势在于模仿自然主义的错误的行为的能力，让实验者观察如何在生产中遇到错误，是用来重新调整电机输出。 。为调查感觉控制的神经基础和可塑性8,9，的鸣鸟提供了一个很好的动物模型</sup&gt;。歌鸟的大脑提供了一个良好定义的电路，在宋学的领域，因为需要在空间上分开歌曲生产的要求，和神经记录和病变的研究取得了显着的进展如何理解大脑不同区域对声音的行为9-12 。然而，由于缺乏一个自然纠错模式 - 在一个已知的声学参数的扰动被实验者，然后纠正的女歌手 - 已很难理解声乐学习相关的计算或不同的神经回路的元素如何贡献校正声乐错误13。 这里描述的技术给出实验者鸟鸣听觉反馈中的错误的精确的控制，允许引入任意的感觉，可用于驱动发声学习错误。在线的声音处理设备用于引入公知的扰动声学的歌曲，和一个小型化的耳机设备是用来取代一个女歌手的自然听觉反馈的扰动信号的实时。我们已经用这种范式干扰在成年鸣禽听觉反馈的基本频率（音调），提供首次证明了成年鸟使用错误修正14，保持人声的表现。本协议可用于实现宽范围的感觉反馈扰动（包括但不限于音高变化）调查的计算和神经电生理基础的声乐学习。

图6示出了一个代表性的实验上执行的成年孟加拉语燕雀。在此，耳机系统被用来增加一个半音（十二分之一的一个八度，相当于约6％的绝对频率的变化）的听觉反馈的间距16天。这种操作导致的所有歌曲音节的音高（彩色线条）在逐渐减少。这种变化导致减少错误所经历的鸟（虚线），这表明鸟的依赖听觉反馈纠正明显的声乐错误的听觉在声乐电机程序。后第16天，当音调移位除去歌曲的间距最终回到基线。 在图6中所示的数据是典型的，因为它们反映不完全适应。在这里，虽然听觉反馈的间距错开1.0个半音，鸟改变他的歌声的间距只有约0.4扫描电镜itones。跨物种和系统的，不完整的适应是虚拟的反馈将被用来干扰一个单一的感官5,15规范，并在目前的模式可能反映了部分依赖在nonauditory信号（ 如本体感觉）鸣禽评估其正在进行的声乐表演。 <img alt="图1" src="/files/ftp_upload/50027/50027fig1.jpg" /> 图1。耳机框架组件。交叉开关组件。连接螺钉，横杆，六角螺母，注射器针头（红色），使用环氧系粘合剂（蓝色），如图所示。每个螺钉的尖端，与矿物油（绿色），以防止环氧树脂粘结的螺丝。耳塞式组装。安装后，气缸，泡沫垫（橙色）与环氧树脂。配件耳机框架。在手术前，通过耳塞的的线程stereotax earbars（黑色）。附加横梁头骨用环氧树脂或牙科丙烯酸（蓝色），然后连接耳塞使用鳄鱼夹的横梁，对鸟的头部轻轻按压泡沫垫。左，侧图的交叉鸟的头部和耳机上的定位。右，耳机帧后，连接到所述颅骨的六角螺母组件的侧视图，已被移除通过拆卸螺钉。 在 B适用于所有面板的标尺。 <img alt="图2" src="/files/ftp_upload/50027/50027fig2.jpg" /> 图2。电子组装。一个。适配器插入吸管尖端的碳纤维气瓶和切割枪头长成废铁一块。环氧树脂，胶水扬声器和耳机的麦克风一起使用一块胶布。分离。胶适配器上扬声器（如图所示），扬声器/耳机麦克风组件。扬声器和耳机麦克风的电线焊接到一个连接器带sOCKET和胶水插座顶端耳机帧。 E的接线板插座上的白点，对准标记。扬声器，耳机麦克风，插座的接线图之间的连接。 <img alt="图3" src="/files/ftp_upload/50027/50027fig3.jpg" /> 图3。铅组装。一个。编造的柔性引线通过焊接四个15厘米长度的导线的连接器条的页眉和编织线拧在一起的一侧上的销。焊接引线的另一端​​连接到换向器的适配器。白点，接线板头对准标记。到接线板插座，耳机喇叭，耳机麦克风，耳机进行改变听觉反馈和记录信号从耳机麦克风的插头插座。对齐点上接线板的插头，插座，以确保正确的连接。 图4。电路的总结。流程图，总结系统的连接。的3个数据信道的记录:（1）未移位从笼中麦克风的信号，（2）的间距移后的信号发送到耳机扬声器，和（3）由耳机麦克风记录的声音波形的副本。前置放大器，麦克风前置放大器，低通滤波器，低通滤波器。 <img alt="图5" src="/files/ftp_upload/50027/50027fig5.jpg" /> 图5。测试系统。 。谱图的声音记录在三个数据通道。每个数据通道显示了三首歌曲音节。颜色代表每一次声频功率。在红色的垂直线所指示的时间的功率谱。请注意，耳机麦克风光谱（绿色）中匹配的峰的峰在日ë移调的（红色），而比未移位（黑）信号，表明到达鸟的耳朵的声音主要是由移动的反馈。 <img alt="图6" src="/files/ftp_upload/50027/50027fig6.jpg" /> 图6。使用耳机系统驱动声乐学习声乐纠错成年孟加拉雀。在耳机系统被用来转移一个半音的音调的听觉反馈向上的彩色线条显示在为期16天的周期（灰盒）的7个不同的歌曲音节的音高变化。黑色实线，意味着在所有的歌曲音节的音高变化。虚线黑线显示的平均螺距误差所经历的主题，在转变的时代。请注意，乐曲的间距的变化的作用是降低的经验丰富的节距误差。球场后移置回零上17天间距的方法基线。第24天的耳机被拆除，然后改为46天，时间间距恢复到基准值。

Watch this Scientific Journal Video about A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds at JoVE.com

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

我们描述了:适合更换一个鸣禽的自然听觉反馈与一个操作的声学信号的微型耳机的设计和组装。在线无害化处理硬件是用来控制歌曲的输出，通过耳机听觉反馈引入实时的错误，电机驱动声乐学习。

一个轻量级的，耳机为基础的系统操作鸣禽听觉反馈

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

a-lightweight-headphones-based-system-for-manipulating-auditory

Research

JoVE Journal

Neuroscience

14.2K 次观看.  Emory University. 我们描述了:适合更换一个鸣禽的自然听觉反馈与一个操作的声学信号的微型耳机的设计和组装。在线无害化处理硬件是用来控制歌曲的输出，通过耳机听觉反馈引入实时的错误，电机驱动声乐学习。

视频: A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

科研

神经科学

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

听觉皮层在成年猫用高场磁共振成像功能成像

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

听觉神经的光遗传学刺激

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

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

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

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

神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合

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

Measuring and Manipulating Functionally Specific Neural Pathways in the Human Motor System with Transcranial Magnetic Stimulation

Understanding interactions between brain areas is important for the study of goal-directed behavior. Functional neuroimaging of brain connectivity has provided important insights into fundamental processes of the brain like cognition, learning, and motor control. However, this approach cannot provide causal evidence for the involvement of brain areas of interest. Transcranial magnetic stimulation (TMS) is a powerful, noninvasive tool for studying the human brain that can overcome this limitation by transiently modifying brain activity. Here, we highlight recent advances using a paired-pulse, dual-site TMS method with two coils that causally probes cortico-cortical interactions in the human motor system during different task contexts. Additionally, we describe a dual-site TMS protocol based on cortical paired associative stimulation (cPAS) that transiently enhances synaptic efficiency in two interconnected brain areas by applying repeated pairs of cortical stimuli with two coils. These methods can provide a better understanding of the mechanisms underlying cognitive-motor function as well as a new perspective on manipulating specific neural pathways in a targeted fashion to modulate brain circuits and improve behavior. This approach may prove to be an effective tool to develop more sophisticated models of brain-behavior relations and improve diagnosis and treatment of many neurological and psychiatric disorders.

This article describes new approaches to measure and strengthen functionally specific neural pathways with transcranial magnetic stimulation. These advanced noninvasive brain stimulation methodologies can provide new opportunities for the understanding of brain-behavior relations and development of new therapies to treat brain disorders.

利用颅内磁刺激测量和操纵人体运动系统中功能特定的神经通路

Understanding interactions between brain areas is important for the study of goal-directed behavior. Functional neuroimaging of brain connectivity has ...

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Here we describe the RatWalker system, built by redesigning the MouseWalker apparatus to accommodate for the increased size and weight of rats. This system uses frustrated total internal reflection (FTIR), high-speed video capture, and open-access analysis software to track and quantify gait parameters.

将RatWalker系统应用于帕金森病遗传大鼠模型中的步态分析

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars ...

Stereocilia Bundle Imaging with Nanoscale Resolution in Live Mammalian Auditory Hair Cells

Inner ear hair cells detect sound-induced displacements and transduce these stimuli into electrical signals in a hair bundle that consists of stereocilia that are arranged in rows of increasing height. When stereocilia are deflected, they tug on tiny (~5 nm in diameter) extracellular tip links interconnecting stereocilia, which convey forces to the mechanosensitive transduction channels. Although mechanotransduction has been studied in live hair cells for decades, the functionally important ultrastructural details of the mechanotransduction machinery at the tips of stereocilia (such as tip link dynamics or transduction-dependent stereocilia remodeling) can still be studied only in dead cells with electron microscopy. Theoretically, scanning probe techniques, such as atomic force microscopy, have enough resolution to visualize the surface of stereocilia. However, independent of imaging mode, even the slightest contact of the atomic force microscopy probe with the stereocilia bundle usually damages the bundle. Here we present a detailed protocol for the hopping probe ion conductance microscopy (HPICM) imaging of live rodent auditory hair cells. This non-contact scanning probe technique allows time lapse imaging of the surface of live cells with a complex topography, like hair cells, with single nanometers resolution and without making physical contact with the sample. The HPICM uses an electrical current passing through the glass nanopipette to detect the cell surface in close vicinity to the pipette, while a 3D-positioning piezoelectric system scans the surface and generates its image. With HPICM, we were able to image stereocilia bundles and the links interconnecting stereocilia in live auditory hair cells for several hours without noticeable damage. We anticipate that the use of HPICM will allow direct exploration of ultrastructural changes in the stereocilia of live hair cells for better understanding of their function.

Here we present a protocol for the Hopping Probe Ion Conductance Microscopy (HPICM), a non-contact scanning probe technique that allows nanoscale imaging of stereocilia bundles in live auditory hair cells.

立体纤毛束成像在活哺乳动物听觉毛细胞中的纳米级分辨率

Inner ear hair cells detect sound-induced displacements and transduce these stimuli into electrical signals in a hair bundle that consists of stereocilia ...

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

Multichannel electrode arrays offer insight into the working brain and serve to elucidate neural processes at the single-cell and circuit levels. Development of these tools is crucial for understanding complex behaviors and cognition and for advancing clinical applications. However, it remains a challenge to densely record from cell populations stably and continuously over long time periods. Many popular electrodes, such as tetrodes and silicon arrays, feature large cross-diameters that produce damage upon insertion and elicit chronic reactive tissue responses associated with neuronal death, hindering the recording of stable, continuous neural activity. In addition, most wire bundles exhibit broad spacing between channels, precluding simultaneous recording from a large number of cells clustered in a small area. The carbon fiber microelectrode arrays described in this protocol offer an accessible solution to these concerns. The study provides a detailed method for fabricating carbon fiber microelectrode arrays that can be used for both acute and chronic recordings in vivo. The physical properties of these electrodes make them ideal for stable and continuous long-term recordings at high cell densities, enabling the researcher to make robust, unambiguous recordings from single units across months.

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

This protocol describes a procedure for constructing carbon fiber microelectrode arrays for chronic and acute in vivo electrophysiological recordings in mouse (Mus musculus) and ferret (Mustela putorius furo) from multiple brain regions. Each step, following the purchase of raw carbon fibers to microelectrode array implantation, is described in detail, with emphasis on microelectrode array construction.

用于慢性和急性体内记录的碳纤维微电极阵列 的 构建和实现

Multichannel electrode arrays offer insight into the working brain and serve to elucidate neural processes at the single-cell and circuit levels. ...

Evaluation of Auditory Brainstem Response in Chicken Hatchlings

The auditory brainstem response (ABR) is an invaluable assay in clinical audiology, non-human animals, and human research. Despite the widespread use of ABRs in measuring auditory neural synchrony and estimating hearing sensitivity in other vertebrate model systems, methods for recording ABRs in the chicken have not been reported in nearly four decades. Chickens provide a robust animal research model because their auditory system is near functional maturation during late embryonic and early hatchling stages. We have demonstrated methods used to elicit one or two-channel ABR recordings using subdermal needle electrode arrays in chicken hatchlings. Regardless of electrode recording configuration (i.e., montage), ABR recordings included 3-4 positive-going peak waveforms within the first 6 ms of a suprathreshold click stimulus. Peak-to-trough waveform amplitudes ranged from 2-11 &#181;V at high-intensity levels, with positive peaks exhibiting expected latency-intensity functions (i.e., increase in latency as a function of decreased intensity). Standardized earphone position was critical for optimal recordings as loose skin can occlude the ear canal, and animal movement can dislodge the stimulus transducer. Peak amplitudes were smaller, and latencies were longer as animal body temperature lowered, supporting the need for maintaining physiological body temperature. For young hatchlings (&#60;3 h post-hatch day 1), thresholds were elevated by ~5 dB, peak latencies increased ~1-2 ms, and peak to trough amplitudes were decreased ~1 &#181;V compared to older hatchlings. This suggests a potential conductive-related issue (i.e., fluid in the middle ear cavity) and should be considered for young hatchlings. Overall, the ABR methods outlined here permit accurate and reproducible recording of in-vivo auditory function in chicken hatchlings that could be applied to different stages of development. Such findings are easily compared to human and mammalian models of hearing loss, aging, or other auditory-related manipulations.

We have used standard auditory brainstem response (ABR) techniques and applied them to hatchling chickens, a precocious avian model for auditory function. The protocol outlines animal preparation and ABR acquisition techniques in detail, with steps that could translate to other avian or rodent models.

鸡幼崽听觉脑干反应的评价

The auditory brainstem response (ABR) is an invaluable assay in clinical audiology, non-human animals, and human research. Despite the widespread use of ...

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Fragile X mental retardation protein (FMRP) is an mRNA-binding protein that regulates local protein translation. FMRP loss or dysfunction leads to aberrant neuronal and synaptic activities in fragile X syndrome (FXS), which is characterized by intellectual disability, sensory abnormalities, and social communication problems. Studies of FMRP function and FXS pathogenesis have primarily been conducted with Fmr1 (the gene encoding FMRP) knockout in transgenic animals. Here we report an in vivo method for determining the cell-autonomous function of FMRP during the period of circuit assembly and synaptic formation using chicken embryos. This method employs stage-, site-, and direction-specific electroporation of a drug-inducible vector system containing Fmr1 small hairpin RNA (shRNA) and an EGFP reporter. With this method, we achieved selective FMRP knockdown in the auditory ganglion (AG) and in one of its brainstem targets, the nucleus magnocellularis (NM), thus providing a component-specific manipulation within the AG-NM circuit. Additionally, the mosaic pattern of the transfection allows within-animal controls and neighboring neuron/fiber comparisons for enhanced reliability and sensitivity in data analyzing. The inducible vector system provides temporal control of gene editing onset to minimize accumulating developmental effects. The combination of these strategies provides an innovative tool to dissect the cell-autonomous function of FMRP in synaptic and circuit development.

Dissecting Cell-Autonomous Function of Fragile X Mental Retardation Protein in an Auditory Circuit by In Ovo Electroporation

Using in ovo electroporation, we devised a method to selectively transfect the auditory inner ear and cochlear nucleus in chicken embryos to achieve a cell-group-specific knockdown of fragile X mental retardation protein during discrete periods of circuit assembly.

一个轻量级的，耳机为基础的系统操作鸣禽听觉反馈

摘要

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此视频中的章节

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

听觉皮层在成年猫用高场磁共振成像功能成像

听觉神经的光遗传学刺激

神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合

利用颅内磁刺激测量和操纵人体运动系统中功能特定的神经通路

将RatWalker系统应用于帕金森病遗传大鼠模型中的步态分析

立体纤毛束成像在活哺乳动物听觉毛细胞中的纳米级分辨率

用于慢性和急性体内记录的碳纤维微电极阵列的构建和实现

鸡幼崽听觉脑干反应的评价

通过产卵电穿孔解剖听觉回路中脆性X智力迟钝蛋白的细胞自主功能

一个轻量级的，耳机为基础的系统操作鸣禽听觉反馈

摘要

探索更多视频

此视频中的章节

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

听觉皮层在成年猫用高场磁共振成像功能成像

听觉神经的光遗传学刺激

神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合

利用颅内磁刺激测量和操纵人体运动系统中功能特定的神经通路

将RatWalker系统应用于帕金森病遗传大鼠模型中的步态分析

立体纤毛束成像在活哺乳动物听觉毛细胞中的纳米级分辨率

用于慢性和急性体内记录的碳纤维微电极阵列 的 构建和实现

鸡幼崽听觉脑干反应的评价

通过产 卵电 穿孔解剖听觉回路中脆性X智力迟钝蛋白的细胞自主功能

用于慢性和急性体内记录的碳纤维微电极阵列的构建和实现

通过产卵电穿孔解剖听觉回路中脆性X智力迟钝蛋白的细胞自主功能