Name: optogenetic stimulation of the auditory nerve
Uploaded: 2014-10-08T15:00:00.000+00:00
Duration: 10 min 53 s
Description: Watch this Scientific Journal Video about Optogenetic Stimulation of the Auditory Nerve at JoVE.com

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>

<ol>
	<li>Rubinstein, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paediatric+cochlear+implantation:+prosthetic+hearing+and+language+development.">Paediatric cochlear implantation: prosthetic hearing and language development.</a> Lancet. 360 (9331), 483-485 (2002).</li><li>Middlebrooks, J. C., Bierer, J. A., Snyder, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+implants:+the+view+from+the+brain.">Cochlear implants: the view from the brain.</a> Current opinion in neurobiology. 15 (4), 488-493 (2005).</li><li>Clark, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multiple-channel+cochlear+implant:+the+interface+between+sound+and+the+central+nervous+system+for+hearing,+speech,+and+language+in+deaf+people-a+personal+perspective.">The multiple-channel cochlear implant: the interface between sound and the central nervous system for hearing, speech, and language in deaf people-a personal perspective.</a> Philosophical transactions of the Royal Society of London. Series B, Biological sciences. 361 (1469), 791-810 (2006).</li><li>Zeng, F. G., Rebscher, S., Harrison, W., Sun, X., Feng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+implants:+system+design,+integration,+and+evaluation.">Cochlear implants: system design, integration, and evaluation.</a> IEEE reviews in biomedical engineering. 1, 115-142 (2008).</li><li>Wilson, B. S., Dorman, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+implants:+a+remarkable+past+and+a+brilliant+future.">Cochlear implants: a remarkable past and a brilliant future.</a> Hearing research. 242 (1-2), 3-21 (2008).</li><li>Moore, D. R., Shannon, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+cochlear+implants:+awakening+the+deafened+brain.">Beyond cochlear implants: awakening the deafened brain.</a> Nature neuroscience. 12 (6), 686-691 (2009).</li><li>Shannon, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multichannel+electrical+stimulation+of+the+auditory+nerve+in+man.+II.+Channel+interaction.">Multichannel electrical stimulation of the auditory nerve in man. II. Channel interaction.</a> Hearing research. 12 (1), 1-16 (1983).</li><li>Fishman, K. E., Shannon, R. V., Slattery, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Speech+recognition+as+a+function+of+the+number+of+electrodes+used+in+the+SPEAK+cochlear+implant+speech+processor.">Speech recognition as a function of the number of electrodes used in the SPEAK cochlear implant speech processor.</a> Journal of speech, language, and hearing research: JSLHR. 40 (5), 1201-1215 (1997).</li><li>Kral, A., Hartmann, R., Mortazavi, D., Klinke, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+resolution+of+cochlear+implants:+the+electrical+field+and+excitation+of+auditory+afferents.">Spatial resolution of cochlear implants: the electrical field and excitation of auditory afferents.</a> Hearing research. 121 (1-2), 11-28 (1998).</li><li>Izzo, A. D., Suh, E., Pathria, J., Walsh, J. T., Whitlon, D. S., Richter, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selectivity+of+neural+stimulation+in+the+auditory+system:+a+comparison+of+optic+and+electric+stimuli.">Selectivity of neural stimulation in the auditory system: a comparison of optic and electric stimuli.</a> Journal of biomedical. 12 (2), 021008 (2007).</li><li>Richter, C. P., Rajguru, S. 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=Spread+of+cochlear+excitation+during+stimulation+with+pulsed+infrared+radiation:+inferior+colliculus+measurements.">Spread of cochlear excitation during stimulation with pulsed infrared radiation: inferior colliculus measurements.</a> Journal of neural engineering. 8 (5), 056006 (2011).</li><li>Teudt, I. U., Maier, H., Richter, C. P., Kral, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acoustic+events+and+&#8220;optophonic&#8221;+cochlear+responses+induced+by+pulsed+near-infrared+laser.">Acoustic events and &#8220;optophonic&#8221; cochlear responses induced by pulsed near-infrared laser.</a> IEEE transactions on bio-medical engineering. 58 (6), 1648-1655 (2011).</li><li>Wang, 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=High-speed+mapping+of+synaptic+connectivity+using+photostimulation+in+Channelrhodopsin-2+transgenic+mice.">High-speed mapping of synaptic connectivity using photostimulation in Channelrhodopsin-2 transgenic mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (19), 8143-8148 (2007).</li><li>Kleinlogel, S., Feldbauer, K., 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=Ultra+light-sensitive+and+fast+neuronal+activation+with+the+Ca2+-permeable+channelrhodopsin+CatCh.">Ultra light-sensitive and fast neuronal activation with the Ca2+-permeable channelrhodopsin CatCh.</a> Nature neuroscience. 14 (4), 513-518 (2011).</li><li>Friesen, L. M., Shannon, R. V., Baskent, D., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Speech+recognition+in+noise+as+a+function+of+the+number+of+spectral+channels:+comparison+of+acoustic+hearing+and+cochlear+implants.">Speech recognition in noise as a function of the number of spectral channels: comparison of acoustic hearing and cochlear implants.</a> The Journal of the Acoustical Society of America. 110 (2), 1150-1163 (2001).</li><li>Donaldson, G. S., Kreft, H. A., Litvak, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Place-pitch+discrimination+of+single-+versus+dual-electrode+stimuli+by+cochlear+implant+users+(L).">Place-pitch discrimination of single- versus dual-electrode stimuli by cochlear implant users (L).</a> The Journal of the Acoustical Society of America. 118 (2), 623-626 (2005).</li><li>Srinivasan, A. G., Shannon, R. V., Landsberger, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+virtual+channel+discrimination+in+a+multi-channel+context.">Improving virtual channel discrimination in a multi-channel context.</a> Hearing research. 286 (1-2), 19-29 (2012).</li><li>Zeng, F. G., 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=Speech+dynamic+range+and+its+effect+on+cochlear+implant+performance.">Speech dynamic range and its effect on cochlear implant performance.</a> The Journal of the Acoustical Society of America. 111 (1 Pt 1), 377-386 (2002).</li><li>Matic, A. I., Walsh, J. T., Richter, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+extent+of+cochlear+infrared+neural+stimulation+determined+by+tone-on-light+masking.">Spatial extent of cochlear infrared neural stimulation determined by tone-on-light masking.</a> Journal of biomedical. 16 (11), 118002 (2011).</li><li>Verma, R., Guex, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+responses+to+electric+and+infrared+neural+stimulation+of+the+rat+cochlear+nucleus.">Auditory responses to electric and infrared neural stimulation of the rat cochlear nucleus.</a> Hearing research. 310, 69-75 (2014).</li><li>Fenno, L., Yizhar, O., 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=The+development+and+application+of+optogenetics.">The development and application of optogenetics.</a> Annual review of neuroscience. 34, 389-412 (2011).</li><li>Hegemann, P., Nagel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+channelrhodopsins+to+optogenetics.">From channelrhodopsins to optogenetics.</a> EMBO molecular medicine. 5 (2), 173-176 (2013).</li><li>Packer, A. M., Roska, B., H&#228;usser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+neurons+and+photons+for+optogenetics.">Targeting neurons and photons for optogenetics.</a> Nature neuroscience. 16 (7), 805-815 (2013).</li><li>Nagel, G., 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=Channelrhodopsin-2,+a+directly+light-gated+cation-selective+membrane+channel.">Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (24), 13940-13945 (2003).</li><li>Nagel, G., Szellas, T., Kateriya, S., Adeishvili, N., Hegemann, P., Bamberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Channelrhodopsins:+directly+light-gated+cation+channels.">Channelrhodopsins: directly light-gated cation channels.</a> Biochemical Society transactions. 33 (Pt 4), 863-866 (2005).</li><li>Nagel, G., Brauner, M., Liewald, J. F., Adeishvili, N., Bamberg, E., Gottschalk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+activation+of+channelrhodopsin-2+in+excitable+cells+of+Caenorhabditis+elegans+triggers+rapid+behavioral+responses.">Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses.</a> Current biology: CB. 15 (24), 2279-2284 (2005).</li><li>Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., 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=Millisecond-timescale,+genetically+targeted+optical+control+of+neural+activity.">Millisecond-timescale, genetically targeted optical control of neural activity.</a> Nature neuroscience. 8 (9), 1263-1268 (2005).</li><li>Bamann, C., Kirsch, T., Nagel, G., Bamberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+characteristics+of+the+photocycle+of+channelrhodopsin-2+and+its+implication+for+channel+function.">Spectral characteristics of the photocycle of channelrhodopsin-2 and its implication for channel function.</a> Journal of molecular biology. 375 (3), 686-694 (2008).</li><li>Ritter, E., Stehfest, K., Berndt, A., Hegemann, P., Bartl, F. 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><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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Changchun New Industries</td><td>MLL-III473</td><td>100 mW solid state 473 nm laser</td></tr><tr><td>Laser driver&amp;nbsp;</td><td>Changchun New Industries</td><td>DPSSL MLL 100 mW</td><td>TTL operated laser driver</td></tr><tr><td>250 &amp;micro;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 &amp;amp; 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 &amp;micro;LED</td><td>Cree</td><td>C470UT200</td><td>It is necessary to build several &amp;mu;LED devices because easily get damaged or the isolation is not good enough</td></tr><tr><td>TDT System&amp;nbsp;</td><td>Tucker-Davis Technologies</td><td>RZ6-A-P1</td><td>It can be used any system for stimulus generation&amp;nbsp; presentation and data acquisition</td></tr><tr><td>Single-shank, 16-channel silicon probe</td><td>Neuronexus</td><td>a1x16-5mm-100-177-CM16LP&amp;nbsp;</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; &lt;em&gt;e.g.&lt;/em&gt; Fine Science Tools</td><td>19007-07</td><td></td></tr><tr><td>Self tapping bone screw</td><td>any commercial; &lt;em&gt;e.g.&lt;/em&gt; Fine Science Tools</td><td>19010-10</td><td>Reference screw</td></tr><tr><td>Micromanipulator</td><td>any commercial; &lt;em&gt;e.g.&lt;/em&gt; 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

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

基因转移到鸡听觉器官通过<em&gt;在卵</em&gt;微电

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

科研

发育生物学

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

Canalostomy 为成人和新生小鼠内耳局部药物传递的外科治疗方法

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

遗传学

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 ...

神经科学

Behavioral Determination of Stimulus Pair Discrimination of Auditory Acoustic and Electrical Stimuli Using a Classical Conditioning and Heart-rate Approach

Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural auditory prostheses, such as auditory brainstem implants1-3 and auditory midbrain implants4,5. While acute experiments can give initial insight to the effectiveness of the implant, testing the chronically implanted and awake animals provides the advantage of examining the psychophysical properties of the sensations induced using implanted devices6,7.
Several techniques such as reward-based operant conditioning6-8, conditioned avoidance9-11, or classical fear conditioning12 have been used to provide behavioral confirmation of detection of a relevant stimulus attribute. Selection of a technique involves balancing aspects including time efficiency (often poor in reward-based approaches), the ability to test a plurality of stimulus attributes simultaneously (limited in conditioned avoidance), and measure reliability of repeated stimuli (a potential constraint when physiological measures are employed).
Here, a classical fear conditioning behavioral method is presented which may be used to simultaneously test both detection of a stimulus, and discrimination between two stimuli. Heart-rate is used as a measure of fear response, which reduces or eliminates the requirement for time-consuming video coding for freeze behaviour or other such measures (although such measures could be included to provide convergent evidence). Animals were conditioned using these techniques in three 2-hour conditioning sessions, each providing 48 stimulus trials. Subsequent 48-trial testing sessions were then used to test for detection of each stimulus in presented pairs, and test discrimination between the member stimuli of each pair. 
This behavioral method is presented in the context of its utilisation in auditory prosthetic research. The implantation of electrocardiogram telemetry devices is shown. Subsequent implantation of brain electrodes into the Cochlear Nucleus, guided by the monitoring of neural responses to acoustic stimuli, and the fixation of the electrode into place for chronic use is likewise shown.

The application of a classical fear conditioning behavioral paradigm for auditory prosthetic research in rats is described. This paradigm provides a mechanism for identifying both detection of, and discrimination between, distinct acoustic and electrical stimuli using heart-rate as an outcome measure.

使用传统的空调和心率的方法刺激听觉声学和电刺激对歧视行为的测定

Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural ...

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 ...

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. ...

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of hearing. Improvement of the implant technology is needed if CI users are to perceive music and perform in more natural environments, such as hearing out a voice with competing talkers, reflections, and other sounds. Such improvement requires experimental animals to better understand the mechanisms of electric stimulation in the cochlea and its responses in the whole auditory system. The mouse is an increasingly attractive model due to the many genetic models available. However, the limited use of this species as a CI model is mainly due to the difficulty of implanting small electrode arrays. More details about the surgical procedure are therefore of great interest to expand the use of mice in CI research.
In this report, we describe in detail the protocol for acute deafening and cochlear implantation of an electrode array in the C57BL/6 mouse strain. We demonstrate the functional efficacy of this procedure with electrically-evoked auditory brainstem response (eABR) and show examples of facial nerve stimulation. Finally, we also discuss the importance of including a deafening procedure when using a normally hearing animal. This mouse model provides a powerful opportunity to study genetic and neurobiological mechanisms that would be of relevance for CI users.

Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem response.

c57bl6 小鼠耳蜗植入术与电唤起脑干反应记录

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of ...

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect distinct cellular and network components of these circuits remains unclear. Ex vivo brain slices provide a means by which investigators may probe discrete components of complex networks and disentangle potential mechanisms underlying the effects of volatile anesthetics on evoked responses. To isolate potential cell type- and pathway-specific drug effects in brain slices, investigators must be able to independently activate afferent fiber pathways, identify non-overlapping populations of cells, and apply volatile anesthetics to the tissue in aqueous solution. In this protocol, methods to measure optogenetically-evoked responses to two independent afferent pathways to neocortex in ex vivo brain slices are described. Extracellular responses are recorded to assay network activity and targeted whole-cell patch clamp recordings are conducted in somatostatin- and parvalbumin-positive interneurons. Delivery of physiologically relevant concentrations of isoflurane via artificial cerebral spinal fluid to modulate cellular and network responses is described.

Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane.

脑切片中传入通路的光遗传学激活和挥发性麻醉剂对反应的调节

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

体外楔形切片准备模拟在Vivvo神经元电路连接

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...

<meta charset="utf8"></head><body>电声刺激中长电极的听力保护

Enhanced Cochlear Coverage and Hearing Preservation in High-Frequency Hearing Loss via Electric Acoustic Stimulation with Longer Electrode

Electric-acoustic stimulation (EAS) is a promising treatment to improve hearing ability in patients with high-frequency hearing loss (HL). In EAS surgeries, shorter electrodes have been preferred to avoid the presence of an electrode covering the residual hearing region. However, our earlier studies showed that EAS with longer electrodes (28 mm) could preserve acoustic hearing. Additionally, we reported that the hearing preservation (HP) scores were independent of the length of the inserted electrodes, consistent with the systematic review. As most EAS patients gradually lose residual hearing over time due to the natural course of HL, in these cases, providing broader cochlear coverage using longer electrodes was beneficial toward better place-pitch matching. In addition to preparing for the deterioration in&#160;hearing in&#160;the future, EAS with longer electrodes could offer various types of map strategies. Herein, we show the pre-, intra-, and post-procedures for EAS surgery. Appropriate preoperative evaluation, less invasive surgery, flexible lateral-wall electrodes, and steroid administration resulted in good HP following EAS with longer electrodes.

<meta charset="utf8"></head><body>作者聚焦:使用长电极优化 EAS 以增强人工耳蜗覆盖率和听力保护

Electric acoustic stimulation (EAS) with longer electrodes can offer broader cochlear coverage and various types of maps in cases of high-frequency hearing loss. Combining less invasive surgery, flexible lateral-wall electrodes, and steroid administration permits deeper insertion with little or no surgical trauma, resulting in good preservation of hearing.

听觉神经的光遗传学刺激

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