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）。

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<ol><li>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）。 </li><li>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化。 </li><li>对于机械稳定性和电绝缘性，通过一个薄的玻璃毛细管漏斗的电线，使用1个外径硼硅酸盐毛细管和用环氧树脂密封的交界处（ 图1D），使得所述毛细管可安装在一个机械手。 注：t这里有一个权衡使得胶囊够厚，以获得良好的电气隔离（避免刺激文物），但足够薄，让LED的自由移动的肺大泡内，以将其定位到内耳开窗。这是一个很好的做法，准备一个以上的LED。 </li><li>确认良好的电隔离，首先准备陪替氏培养皿用0.9％NaCl和浸3裸端的铜线到液体中。这是为了模拟与ABR电极的动物。将电线连接到ABR放大器，并从那里来的示波器。 </li><li>然后，发光二极管连接到刺激器和浸在该溶液中，直到它被完全覆盖。驱动LED的脉冲（见步骤3.1.1）的最大允许电流的时间超过20分钟，并检查是否有刺激的文物。如果他们出现，放弃了LED，并尝试一个新的。 </li></ol> 2，手术过程<ol><li>使用4-10周龄的转基因小鼠表达的ChR2在Thy1.2子（9号线在王某等人13）。操纵动物进行手术，一定要使用干净，很好认的外科手术工具。 </li><li>麻醉动物用腹腔注射乌拉坦（1.32毫克/千克），甲苯噻嗪（5毫克/千克）的混合物，和丁丙诺啡0.1微克/克稀释在0.9％无菌氯化钠溶液，然后将鼠标上的加热板用于保持体温度在37℃。与此剂量，麻醉通常持续整个实验方案。 <ol><li>开始后最初的麻醉诱导检查觉醒的缩足反射的迹象10分钟。的情况下的动作，应用维持剂量氨基甲酸酯（0.66毫克/千克）和丁丙诺啡（0.05微克/克）稀释在0.9％无菌氯化钠溶液（不含甲苯噻嗪）的。再次检查缩足反射并继续仅在反射不存在。 </li></ol></li><li>小心刮耳后区，准备切口。使用耳的方法来达到大疱（含气中耳腔）。 <ol><li>用微型镊子仔细解剖和/或删除颈部肌肉scalenes一部分， 例如，颈阔肌，胸锁乳突肌和。 </li><li>发现和揭露泡（ 图1A）。识别大疱为骨性结构，其特征球形的泡表面的环表示鼓膜（ 图1A）的插入。使用胰岛素针刚刚低于该环的前端的孔。从那里开始，除去覆盖用细咬骨钳（腐食物）或microforceps的泡在这样一种方式，耳蜗岬暴露（ 图1B）骨。 </li></ol></li><li>对于内耳开窗，轻轻刮掉骨胶囊，打开一个小窗口（耳蜗：〜500-800微米），同时注意保留完整的膜迷路（ 图1B）。在第二耳蜗执行内耳开窗转，因为它是远远不够的镫骨动脉和从顶点。打开顶部可以产生包括耳蜗蜗轴的断裂破裂。 </li><li>对于光纤或μLED植入到鼓阶的圆窗插入，小心地用锋利的手术刀尖端划伤骨利基扩大圆窗龛（叶片数11是一个不错的选择）。请务必使用一个新的刀片或手术刀。要非常小心，因为镫骨动脉的运行速度非常接近圆窗（ 图1B）。 </li></ol> 3，光刺激用两种方法<ol><li>对于transcochlear刺激，使用μLEDs或光纤耦合激光器。 <ol><li>安装毛细管携带μLED上的机械手，小心地将μLED定位到内耳开窗。使用oABR，从电流源（通常为5-40 mA在2.7 V）在1-5赫兹引起由3-10毫秒长的刺激，以优化位置和或装置ientation的μLED的。 </li><li>对于激光刺激，用473 nm的连续波激光器和一个250微米光纤（见材料）。耦合光纤的蓝色激光源的理想情况下提供功率的快速模拟控制（否则使用声光器件或较快的快门来定义的光刺激）。利用显微操作，找出光纤的末端直接到内耳开窗，用牙科水泥（transcochlear刺激）修复它。 </li></ol></li><li>为耳蜗内刺激，通过圆窗进入鼓阶插入纤维。然后用10〜30毫瓦（光纤输出的功率）3-10毫秒的激光脉冲在1-5赫兹征求oABR。 oABR的大振幅使实验者观察oABR示波器上诱发由单一刺激来优化发射器的使用oABR振幅作为读出的位置和方向（μLED或纤维）。 </li><li>一旦良好的oABR已经实现，解决了cyanoacr纤维ylate胶水或牙科水泥，等到胶水固体。 注：这是正常的，观察到了一些液体的耳蜗出来。然而，以避免与水泥的聚合，最好是干起来使用细棉灯芯的区域。 </li></ol> 4，录音oABR的<ol><li>插入耳廓下方针状电极，在顶点和上邻近的腿后面，并将其连接到放大器。 </li><li>放大顶点之间的差异潜力及乳突皮下针。样品在50 kHz和过滤器（1-10,000赫兹）的速度。可视化的差异潜力在示波器上理想的记录设置接近最优化的光刺激的位置。使用信号平均（50-1,000x）和数据存储的计算机以供离线分析。 </li></ol> 5，录音光学诱发局部场电位<ol><li>把动物变成立体定位框架。使用镊子分反弹拉扯皮肤覆盖在颅骨，并用剪刀大致从眉心延伸到这种地步，颈部肌肉附着在头骨进行正中切口。 <ol><li>侧面反映了皮肤。纵向切开骨膜沿矢状缝，并轻轻揉露出颅骨，用棉签将其删除侧面。 </li></ol></li><li>一个自定义的金属板位置上暴露颅骨离开板和lambda的尾端之间大约为1毫米的空间。用直径0.7毫米钻仔细钻一个孔进入顶骨。 </li><li>轻轻地拧在0.85毫米直径的螺杆 - 作为从下丘记录潜能参考（IC） - 持用钳子将螺丝只要螺丝没有拧紧。确保螺丝和硬膜之间的良好接触，但不穿透脑。胶螺丝和金属板到与氰基丙烯酸酯胶的头骨。 </li><li>松开n个埃克的肌肉轻轻地从头骨。拉颈部肌肉的尾端大约1-2毫米。确定矢状窦（SSS）和横窦（TS）的交叉点。如果需要，通过润湿用生理氯化钠溶液的骨增加头骨的透明度。 <ol><li>识别出IC小鼠卧直接尾的同店销售和TS交集。识别IC的位置，慢慢地，仔细地后进行环钻延伸约0.5毫米吻侧为1.5mm尾端到TS和大致3毫米横向。 </li><li>以免伤到SSS和TS，因为这些会妨碍成功的实验。在这里，测试trepanned骨是否已经松动了轻推它。使用镊子慢慢抬离骨。 </li></ol></li><li>用适当的适配器连接多声道阵列和探头到显微。为了防止插入到IC中摧毁了多声道录音阵列，小心地取出硬脑膜用小注射器针头的弯曲末端开始接近骨头的边缘。 <ol><li>从立体框架中取出的动物。固定金属板到一间酒吧，以保持立体定位固定。定位在记录头在集成电路并将其插入下微观控制，使得最上面的信道是在表面处刚刚可见。 </li></ol></li><li>放大的记录阵列和在顶骨的参考螺钉，样品的各个信道之间的差电位在32千赫，低通滤波器（300赫兹），并存储在计算机的脱机分析的速率。 </li><li>所有的动物都应该先以麻醉复苏，依照有关安乐死的准则应予以人道安乐死的过程结束。安乐死的合适方法可包括颈脱位或CO 2吸入。 </li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+light-induced+structural+changes+of+Channelrhodopsin-2+by+UV-visible+and+Fourier+transform+infrared+spectroscopy.">Monitoring light-induced structural changes of Channelrhodopsin-2 by UV-visible and Fourier transform infrared spectroscopy.</a> The Journal of biological chemistry. 283 (50), 35033-35041 (2008).</li><li>Berndt, A., Prigge, M., Gradmann, D., Hegemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+open+states+with+progressive+proton+selectivities+in+the+branched+channelrhodopsin-2+photocycle.">Two open states with progressive proton selectivities in the branched channelrhodopsin-2 photocycle.</a> Biophysical journal. 98 (5), 753-761 (2010).</li><li>Kato, H. E., 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=Crystal+structure+of+the+channelrhodopsin+light-gated+cation+channel.">Crystal structure of the channelrhodopsin light-gated cation channel.</a> Nature. 482 (7385), 369-374 (2012).</li><li>Hegemann, P., M&#246;glich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Channelrhodopsin+engineering+and+exploration+of+new+optogenetic+tools.">Channelrhodopsin engineering and exploration of new optogenetic tools.</a> Nature methods. 8 (1), 39-42 (2011).</li><li>Mattis, J., Tye, K. M., 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=Principles+for+applying+optogenetic+tools+derived+from+direct+comparative+analysis+of+microbial+opsins.">Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins.</a> Nature methods. 9 (2), 159-172 (2012).</li><li>Arenkiel, B. R., Peca, J., 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=In+Vivo+Light-Induced+Activation+of+Neural+Circuitry+in+Transgenic+Mice+Expressing+Channelrhodopsin-2.">In Vivo Light-Induced Activation of Neural Circuitry in Transgenic Mice Expressing Channelrhodopsin-2.</a> Neuron. 54 (2), 205-218 (2007).</li><li>Tomita, H., Sugano, E., 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=Visual+Properties+of+Transgenic+Rats+Harboring+the+Channelrhodopsin-2+Gene+Regulated+by+the+Thy-1.2+Promoter.">Visual Properties of Transgenic Rats Harboring the Channelrhodopsin-2 Gene Regulated by the Thy-1.2 Promoter.</a> PLoS ONE. 4 (11), e7679 (2009).</li><li>Hernandez, V. H., 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=Optogenetic+stimulation+of+the+auditory+pathway.">Optogenetic stimulation of the auditory pathway.</a> The Journal of clinical investigation. 124 (3), 1114-1129 (2014).</li><li>Madisen, L., Mao, T., 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=A+toolbox+of+Cre-dependent+optogenetic+transgenic+mice+for+light-induced+activation+and+silencing.">A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing.</a> Nature Neuroscience. 15 (5), 793-802 (2012).</li><li>Yizhar, O., Fenno, L. E., Davidson, T. J., Mogri, M., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetics+in+neural+systems.">Optogenetics in neural systems.</a> Neuron. 71 (1), 9-34 (2011).</li><li>Hernandez, V. H., 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=Optogenetic+stimulation+of+the+auditory+pathway.">Optogenetic stimulation of the auditory pathway.</a> The Journal of clinical investigation. 124 (3), 1114-1129 (2014).</li><li>Gunaydin, L. A., Yizhar, O., Berndt, A., Sohal, V. S., Deisseroth, K., Hegemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+optogenetic+control.">Ultrafast optogenetic control.</a> Nature neuroscience. 13 (3), 387-392 (2010).</li><li>Klapoetke, N. C., Murata, Y., 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=Independent+optical+excitation+of+distinct+neural+populations.">Independent optical excitation of distinct neural populations.</a> Nature methods. 11 (3), 338-346 (2014).</li></ol>

作者宣称，他们没有竞争的经济利益。

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...

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&gt; 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。

<table border="0" cellpadding="0" cellspacing="0" width="769">
	<tbody>
		<tr>
			<td>Urethane</td>
			<td>Sigma Aldrich</td>
			<td>U2500-100G</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Xylazine HCl</td>
			<td>RXV</td>
			<td></td>
			<td>Sedative and analgesic</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Reckitt Benckiser</td>
			<td></td>
			<td>Analgesic</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td>It is used to hold hard tissue, e.g. bone or materials. Never use them to hold soft delicated tissue&#160;</td>
		</tr>
		<tr>
			<td>Dumont #5 - Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td>Only to be used to hold soft tissue</td>
		</tr>
		<tr>
			<td>Fine Scissors - Sharp</td>
			<td>Fine Science Tools</td>
			<td>14060-09</td>
			<td>To open the skin and help with the muscle dissection</td>
		</tr>
		<tr>
			<td>Lempert Rongeurs&#160;</td>
			<td>Fine Science Tools</td>
			<td>16004-16</td>
			<td>They are very useful to easily remove the bone from the bulla</td>
		</tr>
		<tr>
			<td>473-nm laser&#160;</td>
			<td>Changchun New Industries</td>
			<td>MLL-III473</td>
			<td>100 mW solid state 473 nm laser</td>
		</tr>
		<tr>
			<td>Laser driver&#160;</td>
			<td>Changchun New Industries</td>
			<td>DPSSL MLL 100 mW</td>
			<td>TTL operated laser driver</td>
		</tr>
		<tr>
			<td>250 &#181;m optical fiber</td>
			<td>Any comercial ; e.g. Thorlabs</td>
			<td>M42L05</td>
			<td></td>
		</tr>
		<tr>
			<td>Acousto-optical modulator</td>
			<td>Crystal Technology, Inc.</td>
			<td>PCAOM VIS</td>
			<td>Control the amount of light coupled into the fiber from the laser</td>
		</tr>
		<tr>
			<td>Controller for Acousto-optical modulator</td>
			<td>Crystal Technology, Inc.</td>
			<td>160T1-8SAR-24-0.8</td>
			<td>Control the acousto-optic modulator</td>
		</tr>
		<tr>
			<td>Solo2 laser power &#38; energy meter</td>
			<td>Gentec-EO</td>
			<td></td>
			<td>Used to measure light intensity of the LED and the fiber coupled laser</td>
		</tr>
		<tr>
			<td>Blue &#181;LED</td>
			<td>Cree</td>
			<td>C470UT200</td>
			<td>It is necessary to build several &#956;LED devices because easily get damaged or the isolation is not good enough</td>
		</tr>
		<tr>
			<td>TDT System&#160;</td>
			<td>Tucker-Davis Technologies</td>
			<td>RZ6-A-P1</td>
			<td>It can be used any system for stimulus generation&#160; presentation and data acquisition</td>
		</tr>
		<tr>
			<td>Single-shank, 16-channel silicon probe</td>
			<td>Neuronexus</td>
			<td>a1x16-5mm-100-177-CM16LP&#160;</td>
			<td>These are fragile devises, must be handled carefully and cleaned after use</td>
		</tr>
		<tr>
			<td>Omnidrill</td>
			<td>World Precision Instruments</td>
			<td>503598</td>
			<td>Perform craniotomy for IC recordings and reference screw implantation</td>
		</tr>
		<tr>
			<td>Micro Drill Steel Burrs</td>
			<td>any commercial; e.g. Fine Science Tools</td>
			<td>19007-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Self tapping bone screw</td>
			<td>any commercial; e.g. Fine Science Tools</td>
			<td>19010-10</td>
			<td>Reference screw</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>any commercial; e.g. Luigs+NeumannInVivo Unit Junior 4 axis</td>
			<td></td>
			<td>Positioning of recording probe</td>
		</tr>
	</tbody>
</table>

optogenetic stimulation of the auditory nerve

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。

<html>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。...

Watch this Scientific Journal Video about Optogenetic Stimulation of the Auditory Nerve at JoVE.com

Optogenetic Stimulation of the Auditory Nerve

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。

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 ...

optogenetic-stimulation-of-the-auditory-nerve

Research

JoVE Journal

Neuroscience

14.5K 次观看.  University Medical Center Goettingen. 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。 

视频: Optogenetic Stimulation of the Auditory Nerve

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS：使用的Theta突发协议，以探索个人与脆性X综合征与孤独症的可塑性Mechasnism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

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 ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

光纤植入慢性Optogenetic刺激脑组织

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

Optogenetic刺激的逃避行为<em&gt;果蝇</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

禽流感胚胎听神经听觉神经纤维的选择性跟踪

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

在啮齿动物中枢神经系统的体内光遗传学刺激

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. Few studies have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem, which contains second order neurons of the auditory pathway, is a potential site for gene transfer. In this protocol, a technique for direct and maximal exposure of the murine DCN via a posterior fossa approach is demonstrated. This approach allows for either acute or survival surgery. Following direct visualization of the DCN, a host of experiments are possible, including injection of opsins into the cochlear nucleus and subsequent stimulation by an optical fiber coupled to a blue light laser.&#160;Other neurophysiology experiments, such as electrical stimulation and neural injector tracings are also feasible.&#160;The level of visualization and the duration of stimulation achievable make this approach applicable to a wide range of experiments.

The goal of this protocol is to outline a surgical approach to provide direct access to the dorsal cochlear nucleus in a murine model.

在鼠背耳蜗核的听觉通路的光遗传学刺激直接可视化

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. ...

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

神经肌肉功能的应用经皮神经电刺激评估

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.