Name: immunolabeling and counting ribbon synapses in young adult and aged gerbil cochleae
Uploaded: 2022-04-21T14:15:00
Description: Watch this Scientific Journal Video about Immunolabeling and Counting Ribbon Synapses in Young Adult and Aged Gerbil Cochleae at JoVE.com

作者感谢Lichun Zhang帮助建立了该方法和荧光显微镜服务部门，卡尔·冯·奥西茨基大学奥尔登堡分校，用于使用成像设施。这项研究由德国卓越战略-EXC 2177/1下的德国研究基金会（DFG）资助。

所有协议和程序均由德国下萨克森州有关当局批准，许可证编号为AZ 33.19-42502-04-15/1828和33.19-42502-04-15/1990。该协议适用于蒙古沙鼠（M. unguiculatus）的两性。年轻人是指3-12个月的年龄，而沙鼠被认为是36个月及以上的年龄。如果没有另行说明，可以制备缓冲液和溶液并在冰箱中储存长达数月（4-8°C）。使用前，确保缓冲液和溶液未沉淀。
1. 固定和器官采集
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<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-neuron+labeling+in+the+cat+auditory+nerve.">Single-neuron labeling in the cat auditory nerve.</a> Science. 216 (4551), 1239-1241 (1982).</li><li>Bourien, 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=Contribution+of+auditory+nerve+fibers+to+compound+action+potential+of+the+auditory+nerve.">Contribution of auditory nerve fibers to compound action potential of the auditory nerve.</a> Journal of Neurophysiology. 112 (5), 1025-1039 (2014).</li><li>Sergeyenko, Y., Lall, K., Liberman, M. C., Kujawa, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+cochlear+synaptopathy:+An+early-onset+contributor+to+auditory+functional+decline.">Age-related cochlear synaptopathy: An early-onset contributor to auditory functional decline.</a> The Journal of Neuroscience. 33 (34), 13686-13694 (2013).</li><li>Viana, L. 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=Cochlear+neuropathy+in+human+presbycusis:+Confocal+analysis+of+hidden+hearing+loss+in+post-mortem+tissue.">Cochlear neuropathy in human presbycusis: Confocal analysis of hidden hearing loss in post-mortem tissue.</a> Hearing Research. 327, 78-88 (2015).</li><li>Batrel, C., 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=Mass+potentials+recorded+at+the+round+window+enable+the+detection+of+low+spontaneous+rate+fibers+in+gerbil+auditory+nerve.">Mass potentials recorded at the round window enable the detection of low spontaneous rate fibers in gerbil auditory nerve.</a> PLoS ONE. 12 (1), 0169890 (2017).</li><li>Jeffers, P. W. C., Bourien, J., Diuba, A., Puel, J. -. L., Kujawa, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise-induced+hearing+loss+in+gerbil:+Round+window+assays+of+synapse+loss.">Noise-induced hearing loss in gerbil: Round window assays of synapse loss.</a> Frontiers in Cellular Neuroscience. 15, 699978 (2021).</li><li>Gleich, O., Semmler, P., Strutz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+auditory+thresholds+and+loss+of+ribbon+synapses+at+inner+hair+cells+in+aged+gerbils.">Behavioral auditory thresholds and loss of ribbon synapses at inner hair cells in aged gerbils.</a> Experimental Gerontology. 84, 61-70 (2016).</li><li>Cheal, 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+gerbil:+A+unique+model+for+research+on+aging.">The gerbil: A unique model for research on aging.</a> Experimental Aging Research. 12 (1), 3-21 (1986).</li><li>Gates, G. 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S., Hultcrantz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+degeneration+of+the+organ+of+Corti+in+two+genotypes+of+mice.">Age-related degeneration of the organ of Corti in two genotypes of mice.</a> ORL; Journal for Oto-rhino-laryngology and Its Related Specialties. 56 (2), 61-67 (1994).</li><li>Kobrina, 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=Linking+anatomical+and+physiological+markers+of+auditory+system+degeneration+with+behavioral+hearing+assessments+in+a+mouse+(Mus+musculus)+model+of+age-related+hearing+loss.">Linking anatomical and physiological markers of auditory system degeneration with behavioral hearing assessments in a mouse (Mus musculus) model of age-related hearing loss.</a> Neurobiology of Aging. 96, 87-103 (2020).</li><li>Moreno-Garc&#237;a, A., Kun, A., Calero, O., Medina, M., Calero, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+the+role+of+lipofuscin+in+age-related+neurodegeneration.">An overview of the role of lipofuscin in age-related neurodegeneration.</a> Frontiers in Neuroscience. 12, 464 (2018).</li><li>Jensen, T., Holten-Rossing, H., Svendsen, I., Jacobsen, C., Vainer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+myocardial+tissue+with+digital+autofluorescence+microscopy.">Quantitative analysis of myocardial tissue with digital autofluorescence microscopy.</a> Journal of Pathology Informatics. 7, 15 (2016).</li><li>Kalluri, R., Monges-Hernandez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+gradients+in+the+size+of+inner+hair+cell+ribbons+emerge+before+the+onset+of+hearing+in+rats.">Spatial gradients in the size of inner hair cell ribbons emerge before the onset of hearing in rats.</a> Journal of the Association for Research in Otolaryngology. 18 (3), 399-413 (2017).</li><li>Wu, P. Z., Liberman, L. D., Bennett, K., de Gruttola, V., O'Malley, J. T., Liberman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+neural+degeneration+in+the+human+cochlea:+Evidence+for+hidden+hearing+loss+in+the+aging+ear.">Primary neural degeneration in the human cochlea: Evidence for hidden hearing loss in the aging ear.</a> Neuroscience. 407, 8-20 (2019).</li></ol>

使用该协议中概述的方法，可以从年轻成人和老年沙鼠的耳蜗中免疫标记IHC和突触结构，通过突触前和突触后元素的共定位来识别假定的功能突触，将它们分配给单个IHC，并量化它们的数量，体积和位置。这种方法中使用的抗体还标记外毛细胞（OHCs; myoVIIa）及其突触前带。此外，IHC和OHC免疫标记的可行替代方案是针对乙铁林的抗体，OHCs看起来比IHC暗淡得多。
灌注可以使用两...

与年龄相关的听力损失是世界上最常见的疾病之一，影响着世界上三分之一以上的65岁及以上人口1。其根本原因仍在争论中，并正在积极研究，但可能包括连接内毛细胞（IHC）和传入听觉神经纤维的特殊突触的丧失2。这些带状突触包括突触前结构，其囊泡充满与其相连的神经递质谷氨酸盐，以及突触后α-氨基-3-羟基-5-甲基-4-异噁唑丙酸（AMPA）谷氨酸受体3，4，5。在沙鼠中，约20个传入听觉神经纤维接触一个IHC6，7，8。IHC上面向莫迪奥卢斯的纤维与大突触带相对，而连接IHC柱侧的纤维则面向小突触带（即，在猫9，沙鼠7，豚鼠10和小鼠3，11，12，13，14中）。此外，在沙鼠中，突触前带和突触后谷氨酸斑块的大小正相关7，14。与IHC的双极侧的大带相对的纤维口径小，自发率低，阈值高15。有证据表明，低自发率纤维比位于IHC15柱侧的高自发性低阈值纤维更容易受到噪声暴露10和耳毒性药物16的影响。
带状突触的丧失是耳蜗神经年龄相关性听力损失中最早的退行性事件，而螺旋神经节细胞及其传入听觉神经纤维的丧失则落后于17，18。电生理相关性包括听觉脑干反应17 和复合动作电位8的记录;然而，这些并不反映突触丢失的微妙之处，因为低自发率纤维不有助于这些措施16。更有希望的电生理学指标是质量电位衍生的神经指数19 和周震颤时间响应20。然而，只有当动物除了听觉神经纤维丧失之外没有其他耳蜗病理影响剩余听觉神经纤维的活动时，这些才是可靠的8。此外，沙鼠的行为评估阈值与突触数21无关。因此，只有通过直接检查耳蜗组织，才能可靠地定量存活的带状突触，从而确定功能性听觉神经纤维的数量。
蒙古沙鼠（Meriones unguiculatus）是研究与年龄相关的听力损失的合适动物模型。它的寿命短，具有与人类相似的低频听力，易于维护，并且与与年龄相关的听力损失相关的人类病理学表现出相似性2，22，23，24。沙鼠在达到36个月大时被认为是衰老的，这接近其平均寿命的结束22。重要的是，在安静环境中饲养和老化的沙鼠中已经证明了与年龄相关的带状突触丧失8，21。 
在这里，提出了一种免疫标记，解剖和分析不同年龄的沙鼠（从年轻人到老年人）的耳蜗的方案。使用针对前突触 （CtBP2）、突触后谷氨酸受体贴片 （GluA2） 和 IHC （myoVIIa） 成分的抗体。应用自发荧光淬灭剂，减少老化耳蜗的背景，使荧光信号完好无损。此外，还描述了如何解剖耳蜗以检查感觉上皮和纹状血管。测量耳蜗长度是为了能够选择与特定最佳频率相对应的不同耳蜗位置25。突触数的定量是使用免费软件ImageJ26进行的。使用Matlab中编写的软件自定义来执行单个HC中突触体积和位置的额外定量。该软件不公开，因为作者缺乏提供专业文档和支持的资源。

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	<tbody>
		<tr>
			<td>Albumin Fraction V biotin-free</td>
			<td>Carl Roth</td>
			<td>0163.2</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CtBP2 (IgG1 monoclonal mouse)</td>
			<td>BD Biosciences, Eysins</td>
			<td>612044</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-GluA2 (IgG2a monoclonal mouse)</td>
			<td>Millipore</td>
			<td>MAB39</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse (IgG1)-AF 488</td>
			<td>Molecular Probes Inc.</td>
			<td>A21121</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-MyosinVIIa (IgG polyclonal rabbit)</td>
			<td>Proteus Biosciences</td>
			<td>25e6790</td>
			<td></td>
		</tr>
		<tr>
			<td>Blade Holder &#38; Breaker - Flat Jaws</td>
			<td>Fine Science Tools</td>
			<td>10052-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Bonn Artery Scissors - Ball Tip</td>
			<td>Fine Science Tools</td>
			<td>14086-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip thickness 1.5H, 24 x 60 mm</td>
			<td>Carl Roth</td>
			<td>LH26.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Surgical Blade</td>
			<td>Henry Schein</td>
			<td>0473</td>
			<td></td>
		</tr>
		<tr>
			<td>donkey anti-rabbit (IgG)-AF647</td>
			<td>Life Technologies-Molecular Probes</td>
			<td>A-31573</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 - Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5SF Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, absolute 99.8%</td>
			<td>Fisher Scientific</td>
			<td>12468750</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Carl Roth</td>
			<td>8040.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Feather Double Edge Blade</td>
			<td>PLANO</td>
			<td>112-9</td>
			<td></td>
		</tr>
		<tr>
			<td>G19 Cannula</td>
			<td>Henry Schein</td>
			<td>9003633</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti-mouse (IgG2a)-AF568</td>
			<td>Invitrogen</td>
			<td>A-21134</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Ratiopharm</td>
			<td>N68542.04</td>
			<td></td>
		</tr>
		<tr>
			<td>Huygens Essentials</td>
			<td>Scientific Volume Imaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>Fiji</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Immersol, Immersion oil 518F</td>
			<td>Carl Zeiss</td>
			<td>10539438</td>
			<td></td>
		</tr>
		<tr>
			<td>Intrafix Primeline Classic, 150 cm (mit Datamatrix Code auf der Sterilverpackung)</td>
			<td>Braun</td>
			<td>4062957E</td>
			<td></td>
		</tr>
		<tr>
			<td>ISM596D</td>
			<td>Ismatec</td>
			<td></td>
			<td>peristaltic pump</td>
		</tr>
		<tr>
			<td>KL 1600 LED</td>
			<td>Schott</td>
			<td>150.600</td>
			<td>light source for stereomicroscope</td>
		</tr>
		<tr>
			<td>Leica Application suite X</td>
			<td>Leica Microsystem CMS GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica TCS SP8 system</td>
			<td>Leica Microsystem CMS GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>The Mathworks Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mayo Scissors Tungston Carbide ToghCut</td>
			<td>Fine Science Tools</td>
			<td>14512-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-100 Orbital-Genie</td>
			<td>Scientific Industries</td>
			<td>SI-M100</td>
			<td>for use in cold environment</td>
		</tr>
		<tr>
			<td>Narcoren (pentobarbital)</td>
			<td>Boehringer Ingelheim Vetmedica GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ni-Ei</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements</td>
			<td>Nikon Europe B.V.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Carl Roth</td>
			<td>0335.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish without vents</td>
			<td>Avantor VWR</td>
			<td>390-1375</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium phosphate</td>
			<td>AppliChem</td>
			<td>A1046</td>
			<td></td>
		</tr>
		<tr>
			<td>Monopotassium phosphate</td>
			<td>Carl Roth</td>
			<td>3904.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Carl Roth</td>
			<td>6781.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma Aldrich</td>
			<td>31434-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw Cap Containers</td>
			<td>Sarstedt</td>
			<td>75.562.300</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Carl Roth</td>
			<td>K305.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Student Adson Forceps</td>
			<td>Fine Science Tools</td>
			<td>91106-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Student Halsted-Mosquito Hemostat</td>
			<td>Fine Science Tools</td>
			<td>91308-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Adhesion Microscope Slides</td>
			<td>Epredia</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton&#160; X</td>
			<td>Carl Roth</td>
			<td>3051.2</td>
			<td></td>
		</tr>
		<tr>
			<td>TrueBlack Lipofuscin Autofluorescence Quencher</td>
			<td>Biotium</td>
			<td>23007</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors, 3mm</td>
			<td>Fine Science Tools</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield Antifade Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrax VXR basic</td>
			<td>IKA</td>
			<td>0002819000</td>
			<td></td>
		</tr>
		<tr>
			<td>VX 7 Dish attachment for Vibrax VXR basic</td>
			<td>IKA</td>
			<td>953300</td>
			<td></td>
		</tr>
		<tr>
			<td>Wild TYP 355110 (Stereomicroscope)</td>
			<td>Wild Heerbrugg</td>
			<td></td>
			<td>not available anymore</td>
		</tr>
	</tbody>
</table>

immunolabeling and counting ribbon synapses in young adult and aged gerbil cochleae

连接内部毛细胞和传入听觉神经纤维的带状突触的缺失被认为是年龄相关性听力损失的原因之一。检测带状突触丢失的最常见方法是免疫标记，因为它允许从单个耳蜗中的多个视点进行定量采样。然而，感兴趣的结构深埋在骨耳蜗内。沙鼠被用作年龄相关听力损失的动物模型。在这里，描述了固定的常规方案，免疫标记沙鼠耳蜗整个支架，共聚焦成像以及量化带状突触数量和体积。此外，还强调了与从有价值的老年人那里获得优质材料相关的特殊挑战。 
沙鼠被安乐死，要么心血管灌注，要么它们的鼓膜大疱被小心地从头骨中解剖出来。耳蜗在顶点和基部打开，并直接转移到固定剂中。无论初始方法如何，耳蜗都会被后固定并随后脱钙。然后用针对突触前和突触后结构和毛细胞的一抗标记组织。接下来，将耳蜗与二级荧光标记抗体一起孵育，这些抗体对各自的主要抗体具有特异性。然后用自发荧光淬灭剂处理老年沙鼠的耳蜗，以减少老年动物组织通常实质性的背景荧光。 
最后，将耳蜗解剖成6-11节。重建整个人工耳蜗长度，以便可靠地确定个体之间的特定人工耳蜗位置。按顺序获取的共聚焦图像堆栈有助于可视化所选位置的毛细胞和突触。共聚焦堆栈被解卷积，突触要么使用ImageJ手动计数，要么使用Matlab中定制的图像分析程序对突触结构进行更广泛的量化。

耳蜗要么在心血管灌注后用整只动物的固定剂收获，要么在安乐死动物并浸泡固定后迅速解剖。使用后一种方法，IHC在解剖过程中保持在原位，而在灌注不成功且因此组织固定不足的情况下，感觉上皮经常被破坏。请注意，作者遇到的情况是，经心灌注后耳蜗固定不足，而大脑固定仍然足够。通过打开进入耳蜗顶点和基底的孔，并通过浸入4%PFA浸入耳蜗2天来固定耳蜗，仍然可以保存来自下灌注的?...

Watch this Scientific Journal Video about Immunolabeling and Counting Ribbon Synapses in Young Adult and Aged Gerbil Cochleae at JoVE.com

Immunolabeling and Counting Ribbon Synapses in Young Adult and Aged Gerbil Cochleae

提出了一种通过免疫标记传入突触结构和毛细胞，在老年组织中淬灭自发荧光，解剖和估计耳蜗的长度以及量化通过共聚焦成像获得的图像堆栈中的突触来处理年轻成人和老年沙鼠耳蜗的方案。

年轻成人和老年沙鼠耳蜗的免疫标记和计数带状突触

The loss of ribbon synapses connecting inner hair cells and afferent auditory nerve fibers is assumed to be one cause of age-related hearing loss. The ...

该协议能够评估所有年龄段沙鼠耳蜗中的突触完整性。这对于听力问题的基础研究非常重要，特别是由于衰老。该方案相对较快，可从耳蜗沿线的几个位置生成定量数据。此外，它通过使用自发荧光淬灭剂来解决组织老化的特定问题。突触变性是耳蜗功能丧失的最早迹象。量化它是研究开发突触病非侵入性诊断工具的重要工具。人工耳蜗解剖是需要练习的最关键的部分，所以最好从非实验性耳蜗开始。首先去除大疱。用剪刀切开骨头或用镊子开裂骨头，然后用镊子将其取出以到达耳蜗。切除踝、胯骨和半规管。小心地取下覆盖耳蜗顶端的骨头，并用镊子划伤耳蜗骨，在顶点和基底转弯处打出小孔，或者在两个位置小心地戳孔。立即将大疱转移到至少50毫升的冷固定剂中。要将耳蜗切成两半，请将一块长度超过耳蜗盘绕长度的剃须刀片放入刀片架中。然后，将耳蜗置于体视显微镜下的培养皿中。用剃须刀片或细剪刀切掉多余的组织。用细镊子将耳蜗固定到位，并沿着镊子切成两半。通过将自身荧光淬灭剂与70%乙醇混合来制备5%的溶液。将大肠杆菌置于该溶液中，并在室温下在摇床上孵育一分钟，然后用一毫升PBS洗涤三次，持续五分钟。为了解剖耳蜗，收集两个细镊子，超细镊子，叶片，弹簧剪刀，刀片支架，剃须刀片，并用PBS填充聚苯乙烯培养皿及其盖子。从剃须刀刀片上掰开碎片，以获得两到四毫米的切割表面。将耳蜗切成两半，如组织制备部分所示。用镊子小心地固定耳蜗的一半，使切割边缘朝上。使用细弹簧剪刀切除覆盖顶端的螺旋骨上方的耳蜗骨。然后，使用剪刀通过切开莫迪奥勒和听觉神经以及 Scala 前庭来隔离顶端转弯。在上面的耳蜗骨的每一侧进行两次切口，或者如果不需要进一步评估血管纹，则直接通过中间转弯的纹状血管。使用剃须刀片分离耳蜗碎片。或者，在皮质器官和血管纹之间切割。将纹状血管连接到螺旋韧带，并使用镊子去除脱钙的骨。接下来，将人工耳蜗转移到装有PBS的培养皿盖中。去除柱子和磨砂面上多余的组织，以便整个支架稍后将尽可能平坦地放在载玻片上。大的耳蜗碎片，特别是那些来自基底转弯的耳蜗碎片，应该被切成两块。用超细镊子小心地去除胸膜。将耳蜗碎片放在滑轨上，放入一滴安装介质中，确保Corti器官朝上。将人工耳蜗移入矢状面，并在靠近内毛细胞的地方寻找螺旋边缘的内陷。螺旋边缘的这种内陷在中耳蜗和基底耳蜗碎片中可以更清楚地看到。通过重复这些步骤将整个耳蜗和整个血管纹转移到显微镜载玻片上。盖上滑轨，并在边缘周围添加黑色指甲油以进行密封。在黑暗中干燥，并将其储存在四摄氏度。在装有BioVoxxel插件的ImageJ软件中打开反卷积堆栈的副本。通过单击“图像”、“颜色”和“拆分通道”来拆分通道。通过单击“图像”，然后单击“颜色”，然后单击“合并通道”以分配不同的颜色来合并它们。通过单击“图像”将图像转换为 RGB 堆栈，然后转到“颜色”并单击“堆栈到 RGB”。通过单击“图像”，“调整”，然后单击“亮度/对比度”来调整亮度和对比度。使用自动或滑块来确保突触前后结构和内部毛细胞标签与背景不同。使用文本工具标记五个内部毛发单元，方法是单击堆栈中的所需位置。接下来，转到“分析”，然后转到“工具”，然后选择“ROI 管理器”。选中复选框标签以使用数字标记点。放大感兴趣的内部毛细胞。右键单击点工具以选择多点模式。滚动浏览 Z 维度并计算功能性带状突触，这些突触突触前突触突触带状突触突触后谷氨酸贴片并置以识别。选择所有感兴趣的结构后，单击添加 ROI 管理器，然后单击任意名称并选择重命名。使用手动工具，调整图像以完全显示下一个内部毛细胞。从多点工具更改为点工具，以避免向以前存储的数据添加更多点符号。单击下一个内部毛细胞中的感兴趣结构，然后更改为多点工具。继续在ROI管理器中添加功能性色带突触，直到评估所有内部毛细胞。单击 ROI 管理器中的数据集，然后单击“度量”。等待出现列出测量数据点的新窗口，然后单击“文件”和“另存为”以将此列表另存为电子表格。通过在 ROI 管理器中激活“全部显示”来保存图像。单击“展平”以将点标签永久添加到堆栈，然后单击“同意”以通过保存更改来关闭。来自10个月龄的年轻沙鼠耳蜗的Z堆栈投影显示，这里由白色虚线勾勒的肌球蛋白7A标记的内毛细胞与背景形成鲜明对比。突触前的带标有绿色的抗CtBP2二，突触后的谷氨酸贴片标有红色的抗GluA2。来自38个月大的老沙鼠的耳蜗用自发荧光淬灭剂处理，并显示出明显的内部毛细胞，具有清晰可见的突触前和突触后结构。为了测试免疫标记的稳定性，在三个月和29 1/2个月后对来自年轻沙鼠的同一块耳蜗进行成像。尽管信噪比降低，但功能突触是不同的和可量化的。即使切口失败，也要安装剩余的组织片，以便以后最准确地估计整个耳蜗长度。如果先于电生理或心理物理听力测试，然后单独与该协议中的数据相关联，则该协议的数据特别有价值。

Research

JoVE Journal

Neuroscience

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

科研

神经科学

Kinematics and Ground Reaction Force Determination: A Demonstration Quantifying Locomotor Abilities of Young Adult, Middle-aged, and Geriatric Rats

运动学与地面反作用力的测定：一个示范量化青年，中年和老年大鼠的运动能力能力

Behavior, in its broadest definition, can be defined as the motor manifestation of physiologic processes.  As such, all behaviors manifest through the ...

Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats

老年帕金森病大鼠的超声波发声的有针对性的培训

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting ...

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

成年鼠脑铸造三维成像和形态分析

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

亚细胞钙荧光和生物发光成像<sup&gt; 2+</sup&gt;老年海马神经元

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

高分辨率定量免疫膜受体在视网膜带状突触分析

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

结合光遗传学和冷冻断裂免疫标记副本研究在老鼠杏仁核谷氨酸受体的特定投入安排

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea

小鼠耳蜗特定频率区域带状带突起的形态和功能评估

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have ...

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

在果蝇黑色无gaster的Axon死亡期间，对Axon及其突触的形态和功能评估

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our ...