听觉神经的光遗传学刺激
1InnerEarLab, Department of Otolaryngology,University Medical Center Goettingen, 2Bernstein Focus for Neurotechnology,University of Goettingen, 3Auditory Systems Physiology Group, Department of Otolaryngology,University Medical Center Goettingen, 4Center for Nanoscale Microscopy and Molecular Physiology of the Brain,University of Goettingen, 5Department of Chemical, Electronic, and Biomedical Engineering,University of Guanajuato
Summary
人工耳蜗植入（CI）的启用在听取了听神经的直接电刺激。然而，可怜的频率和强度的分辨率限制了听觉与独联体的质量。在这里，我们在小鼠中描述的听觉神经的光遗传学刺激作为听觉的研究和制定未来的独联体的替代策略。
Abstract
螺旋神经节神经元（SGNS）通过人工耳蜗植入（CI）的直接电刺激使开放式的言语理解中的大部分植入聋者1 6。然而，声音编码与当前的证明书有很差的频率和强度的分辨率，由于目前广泛流传的每个电极接触沿着耳蜗7-9的tonotopic轴激活大量SGNS的。光学刺激，提出作为替代的电刺激，有望在空间上更加狭窄活化SGNS的，因此，编码的更高的频率分辨率。在最近几年中，耳蜗的直接红外照明已被用来唤起应答的听觉神经10。尽管如此，它需要更高的能量比电刺激10,11和不确定性仍然是底层机制12。在这里，我们描述了基于光遗传学的方法来刺激SGNS用低强度蓝光，使用转基因小鼠与channelrhodopsin 2（的ChR2）13或的ChR2变爪14的病毒介导的表达的神经元表达。我们使用的微型发光二极管（μLEDs）和光纤耦合激光器通过一个小的人造口（内耳）或圆窗刺激的ChR2表达SGNS。我们测定的反应由光诱发电位（光遗传学听性脑干反应：oABR）头皮录音或从听觉通道微电极记录和声学和电刺激进行比较。
Introduction
根据世界卫生组织，3.6亿人在全球遭受听力损失。在聋者，SGNS独联体的直接电刺激能在大部分1,2,4,5开言语理解。尽管独联体已植入超过20万人，因此是最成功的神经假，声音编码由目前的人工耳蜗驱动是有限的。证明书是由一定数目的电极，其中每一个激活的听觉神经从而绕过尔蒂的功能失调感觉器官在耳蜗Âtonotopic区域基于电刺激。听力正常的听众可以区分2000多个频率，但今天的独联体只使用到12-22频道4。这是由于从各刺激电极7,9，激活大量代表许多不同的声音频率8,15 SGNS的广泛的电流流动。这限制可使用多极的刺激，但在更高的功耗16,17为代价得到改善。它们的输出动态范围的声音强度也有限，一般低于6-20分贝4,18。由于这些原因，提高频率和强度分辨率是用于提高CI性能改善语音识别在嘈杂的环境中，韵律理解和音乐的感知重要目标。
不同的选项来刺激听觉神经的光学刺激。光可以很方便地集中针对一个小SGN人口，并承诺更好的空间约束，提高频率分辨率，并扩大动态范围，从而更好的强度分辨率。事实上，耳蜗刺激红外光表明在动物模型10,11,19良好的频率分辨率。这种刺激的一个缺点是，它需要更高的能量比电刺激<suP> 10,11。此外，关于该法的直接刺激听觉神经元的能力的担忧已经引发12,20。
作为替代红外线刺激，我们采用光遗传学来呈现SGNS光敏感。光遗传学是一种新颖的方法，结合遗传和光学技术，以非侵入性和特异性对照细胞高时间精度（条21-23）。目前最常用的方式使用莱茵衣藻的微生物channelrhodopsin 2（的ChR2）基因的表达和其变体，编码光门控阳离子通道24。的ChR2是7次跨膜螺旋蛋白质，当变换成神经元和由蓝色光激活时，作为非选择性阳离子通道，从而去极化的细胞24 27。的ChR2已被充分表征24,28- 31和许多的变体已经被开发来修改共同行动Ñ 谱，门控和渗透性能32,33。我们工作的目的是建立耳蜗光遗传学的听觉通路的激活。我们注意到，该光遗传学方法来刺激听觉神经需要的螺旋神经节为channelrhodopsin的表达的遗传操作。用小鼠和大鼠的工作允许使用现有的转基因动物13,34,35，提供channelrhodopsin表达沿tonotopic轴线和整个动物36小变异。组合条件基因37与适当的Cre-线提供细胞特异性表达。基因转移到其它动物的螺旋神经节需要使用病毒，如腺伴随病毒也就是在光遗传学38的标准方法，并且我们发现在小鼠36工作良好。基因操作和转基因的不利影响，如IMMU编码外来蛋白质的熊风险的表达NE的反应和/或增殖，破坏状态，甚至死亡基因操作的细胞。对于本演示的目的，我们使用下THY-1启动子13螺旋神经节神经元表达的ChR2基因小鼠光学刺激听觉通路。我们注意到，其他channelrhodopsin变体可以被用于相同的目的，我们证明了使用变爪14的病毒介导的转移进SGNS 39。
虽然人工耳蜗光遗传学，需要遗传操作，它提供了分子调整优化SGN刺激相比，电刺激承诺提高频率和强度分辨率。听觉通路的光遗传学刺激听觉的研究具有很强的针对性。例如，它有望在tonotopy的活性依赖性细化的研究进展发展过程中，在对在声localizat频谱整合需求的分析离子与在中枢听觉系统的频率特异性传入突起之间的相互作用程度的。
Protocol
在这项工作中提出的所有实验进行了由德国法律对实验动物的保护规定的道德标准。哥廷根大学董事会对动物福利和下萨克森州的动物福利办公室批准的实验。 1，准备μLED，刺激的对于μLEDs，首先准备μLED刺激器。使用蓝光LED与200由200微米的活性表面（μLED，见材料表）。 焊接线连接到μLED。然后，封装μLED并用环氧树脂胶的连接。将设备留在一夜之间让环氧固…
Representative Results
一个最佳的内耳开窗是关键的，增加了成功实验的概率， 这意味着该窗口是规则的，小的，并且有内部的耳蜗结构中的无损伤。例如，出血表示血管纹的损坏。一个很好的例子是在图1B。 使用的ChR2-转基因小鼠，的ChR2表达于耳蜗（ 图1C）内的SGNS。蓝色光照明，或者通过μLED或激光时，引发大oABR，这从声ABR（AABR）在振幅和波形不同。…
Discussion
所描述的实验证明了SGNS的光遗传学刺激，并且可以在原则上，也可以用于刺激内和/或外毛细胞，提供视蛋白的表达。这些实验需要极大的耐心和关怀。如之前所提到的，最重要的步骤是一个很好的内耳开窗/圆窗插入所述光源，以及一个适当的位置和方向。
有使用的ChR2时与光遗传学刺激局限性。与光照强度和脉冲持续时间，但我们的情况oABR振幅增大时，减少刺激的速度提高…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作是由德国联邦教育与研究部（伯恩斯坦的焦点神网授予01GQ0810，到T莫泽，和MED-EL德国）的支持;通过中心纳米显微镜和脑分子生理学德国研究基金会（FZT 103，T莫泽），并通过SFB889，为N. Strenzke和T.莫泽）。
Materials
| Urethane | Sigma Aldrich | U2500-100G | Anesthetic |
| Xylazine HCl | RXV | Sedative and analgesic | |
| Buprenorphine | Reckitt Benckiser | Analgesic | |
| Dumont #5 Forceps | Fine Science Tools | 11251-10 | It is used to hold hard tissue, e.g. bone or materials. Never use them to hold soft delicated tissue |
| Dumont #5 – Fine Forceps | Fine Science Tools | 11254-20 | Only to be used to hold soft tissue |
| Fine Scissors – Sharp | Fine Science Tools | 14060-09 | To open the skin and help with the muscle dissection |
| Lempert Rongeurs | Fine Science Tools | 16004-16 | They are very useful to easily remove the bone from the bulla |
| 473-nm laser | Changchun New Industries | MLL-III473 | 100 mW solid state 473 nm laser |
| Laser driver | Changchun New Industries | DPSSL MLL 100 mW | TTL operated laser driver |
| 250 µm optical fiber | Any comercial ; e.g. Thorlabs | M42L05 | |
| Acousto-optical modulator | Crystal Technology, Inc. | PCAOM VIS | Control the amount of light coupled into the fiber from the laser |
| Controller for Acousto-optical modulator | Crystal Technology, Inc. | 160T1-8SAR-24-0.8 | Control the acousto-optic modulator |
| Solo2 laser power & energy meter | Gentec-EO | Used to measure light intensity of the LED and the fiber coupled laser | |
| Blue µLED | Cree | C470UT200 | It is necessary to build several μLED devices because easily get damaged or the isolation is not good enough |
| TDT System | Tucker-Davis Technologies | RZ6-A-P1 | It can be used any system for stimulus generation presentation and data acquisition |
| Single-shank, 16-channel silicon probe | Neuronexus | a1x16-5mm-100-177-CM16LP | These are fragile devises, must be handled carefully and cleaned after use |
| Omnidrill | World Precision Instruments | 503598 | Perform craniotomy for IC recordings and reference screw implantation |
| Micro Drill Steel Burrs | any commercial; e.g. Fine Science Tools | 19007-07 | |
| Self tapping bone screw | any commercial; e.g. Fine Science Tools | 19010-10 | Reference screw |
| Micromanipulator | any commercial; e.g. Luigs+NeumannInVivo Unit Junior 4 axis | Positioning of recording probe |
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Citazione di questo articolo
Hernandez, V. H., Gehrt, A., Jing, Z., Hoch, G., Jeschke, M., Strenzke, N., Moser, T. Optogenetic Stimulation of the Auditory Nerve. J. Vis. Exp. (92), e52069, doi:10.3791/52069 (2014).