Name: morphological and functional evaluation of ribbon synapses at specific frequency regions of the mouse cochlea
Uploaded: 2019-05-10T13:30:00
Description: Watch this Scientific Journal Video about Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea at JoVE.com

这项工作得到了国家自然科学基金(81770997、81771016、81830030)的支持;北京市自然科学基金委员会与北京市教委联合资助项目(KZ201810025040);北京自然科学基金(7174291);中国博士后科学基金会(2016M601067)。

所有程序均按照《NRC/ILAR实验室动物护理和使用指南》(第8版)进行。研究规程经首都医科大学动物护理与使用委员会批准。
1. 动物选择
<ol><li>对于所有实验,使用成年C57BL/6J雄性小鼠(8周大)作为动物模型。 注:C57BL/6J 小鼠携带Cdh23的拼接变体,在听觉系统中表现出加速衰老,在耳蜗的基底转处表现为带状突触损失 40%,在 6 个月大时中间转时损失 10%,随后迅速增?...

<ol>
	<li>Lie, A., Skogstad, M., Johnsen, T. S., Engdahl, B., Tambs, 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+prevalence+of+notched+audiograms+in+a+cross-sectional+study+of+12,055+railway+workers.">The prevalence of notched audiograms in a cross-sectional study of 12,055 railway workers.</a> Ear and Hearing. 36 (3), 86-92 (2015).</li><li>Fettiplace, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+cell+transduction,+tuning,+and+synaptic+transmission+in+the+mammalian+cochlea.">Hair cell transduction, tuning, and synaptic transmission in the mammalian cochlea.</a> Comprehensive Physiology. 7 (4), 1197-1227 (2017).</li><li>Liberman, M. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+physiological+place-frequency+map+of+the+cochlea+in+the+CBA/J+mouse.">A physiological place-frequency map of the cochlea in the CBA/J mouse.</a> Hearing Research. 202 (1-2), 63-73 (2005).</li><li>Viberg, A., Canlon, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+guide+to+plotting+a+cochleogram.">The guide to plotting a cochleogram.</a> Hearing Research. 197 (1-2), 1-10 (2004).</li><li>Paquette, S. T., Gilels, F., White, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise+exposure+modulates+cochlear+inner+hair+cell+ribbon+volumes,+correlating+with+changes+in+auditory+measures+in+the+FVB/nJ+mouse.">Noise exposure modulates cochlear inner hair cell ribbon volumes, correlating with changes in auditory measures in the FVB/nJ mouse.</a> Scientific Reports. 6, 25056 (2016).</li><li>Fernandez, K. A., Jeffers, P. W., 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=Aging+after+noise+exposure:+acceleration+of+cochlear+synaptopathy+in+"recovered"+ears.">Aging after noise exposure: acceleration of cochlear synaptopathy in "recovered" ears.</a> Journal of Neuroscience. 35 (19), 7509-7520 (2015).</li><li>Wichmann, C., Moser, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relating+structure+and+function+of+inner+hair+cell+ribbon+synapses.">Relating structure and function of inner hair cell ribbon synapses.</a> Cell and Tissue Research. 361 (1), 95-114 (2015).</li><li>Matthews, G., Fuchs, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diverse+roles+of+ribbon+synapses+in+sensory+neurotransmission.">The diverse roles of ribbon synapses in sensory neurotransmission.</a> Nature Reviews Neuroscience. 11 (12), 812-822 (2010).</li><li>Liberman, L. D., 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=Postnatal+maturation+of+auditory-nerve+heterogeneity,+as+seen+in+spatial+gradients+of+synapse+morphology+in+the+inner+hair+cell+area.">Postnatal maturation of auditory-nerve heterogeneity, as seen in spatial gradients of synapse morphology in the inner hair cell area.</a> Hearing Research. 339, 12-22 (2016).</li><li>Yang, L., 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=Maximal+number+of+pre-synaptic+ribbons+are+formed+in+cochlear+region+corresponding+to+middle+frequency+in+mice.">Maximal number of pre-synaptic ribbons are formed in cochlear region corresponding to middle frequency in mice.</a> Acta Oto-Laryngologica. 138 (1), 25-30 (2018).</li><li>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=Cochlear+synaptopathy+in+acquired+sensorineural+hearing+loss:+Manifestations+and+mechanisms.">Cochlear synaptopathy in acquired sensorineural hearing loss: Manifestations and mechanisms.</a> Hearing Research. 349, 138-147 (2017).</li><li>Moser, T., Starr, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+neuropathy--neural+and+synaptic+mechanisms.">Auditory neuropathy--neural and synaptic mechanisms.</a> Nature Reviews Neurology. 12 (3), 135-149 (2016).</li><li>Yu, W. 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=A+Gata3-Mafb+transcriptional+network+directs+post-synaptic+differentiation+in+synapses+specialized+for+hearing.">A Gata3-Mafb transcriptional network directs post-synaptic differentiation in synapses specialized for hearing.</a> Elife. 2, 01341 (2013).</li><li>Buniello, 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=Wbp2+is+required+for+normal+glutamatergic+synapses+in+the+cochlea+and+is+crucial+for+hearing.">Wbp2 is required for normal glutamatergic synapses in the cochlea and is crucial for hearing.</a> EMBO Molecular Medicine. 8 (3), 191-207 (2016).</li><li>Gilels, F., Paquette, S. T., Beaulac, H. J., Bullen, A., White, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Severe+hearing+loss+and+outer+hair+cell+death+in+homozygous+Foxo3+knockout+mice+after+moderate+noise+exposure.">Severe hearing loss and outer hair cell death in homozygous Foxo3 knockout mice after moderate noise exposure.</a> Scientific Reports. 7 (1), 1054 (2017).</li><li>Kane, K. L., 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=Genetic+background+effects+on+age-related+hearing+loss+associated+with+Cdh23+variants+in+mice.">Genetic background effects on age-related hearing loss associated with Cdh23 variants in mice.</a> Hearing Research. 283 (1-2), 80-88 (2012).</li><li>Jiang, X. W., Li, X. R., Zhang, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+of+ribbon+synapses+number+of+cochlear+hair+cells+in+C57BL/6J+mice+with+age+(Delta).">Changes of ribbon synapses number of cochlear hair cells in C57BL/6J mice with age (Delta).</a> International Journal of Clinical and Experimental Medicine. 8 (10), 19058-19064 (2015).</li><li>Akil, O., Oursler, A., Fan, K., Lustig, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+auditory+brainstem+response+testing.">Mouse auditory brainstem response testing.</a> BIO-Protocol. 6 (6), (2016).</li><li>Zhou, X., Jen, P. H. -. S., Seburn, K. L., Frankel, W. N., Zheng, Q. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auditory+brainstem+responses+in+10+inbred+strains+of+mice.">Auditory brainstem responses in 10 inbred strains of mice.</a> Brain Research. 1091 (1), 16-26 (2006).</li><li>Montgomery, S. C., Cox, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+mount+dissection+and+immunofluorescence+of+the+adult+mouse+cochlea.">Whole mount dissection and immunofluorescence of the adult mouse cochlea.</a> Journal of Visualized Experiments. (107), (2016).</li><li>Schmitz, F., Konigstorfer, A., Sudhof, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RIBEYE,+a+component+of+synaptic+ribbons:+a+protein's+journey+through+evolution+provides+insight+into+synaptic+ribbon+function.">RIBEYE, a component of synaptic ribbons: a protein's journey through evolution provides insight into synaptic ribbon function.</a> Neuron. 28 (3), 857-872 (2000).</li><li>Suzuki, J., Corfas, G., 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=Round-window+delivery+of+neurotrophin+3+regenerates+cochlear+synapses+after+acoustic+overexposure.">Round-window delivery of neurotrophin 3 regenerates cochlear synapses after acoustic overexposure.</a> Scientific Reports. 6, 24907 (2016).</li><li>Rutherford, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+the+structure+of+inner+ear+ribbon+synapses+with+STED+microscopy.">Resolving the structure of inner ear ribbon synapses with STED microscopy.</a> Synapse. 69 (5), 242-255 (2015).</li><li>Liberman, L. D., 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=Dynamics+of+cochlear+synaptopathy+after+acoustic+overexposure.">Dynamics of cochlear synaptopathy after acoustic overexposure.</a> Journal of the Association for Research in Otolaryngology. 16 (2), 205-219 (2015).</li><li>Gilels, F., Paquette, S. T., Zhang, J., Rahman, I., White, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+of+Foxo3+causes+adult+onset+auditory+neuropathy+and+alters+cochlear+synapse+architecture+in+mice.">Mutation of Foxo3 causes adult onset auditory neuropathy and alters cochlear synapse architecture in mice.</a> Journal of Neuroscience. 33 (47), 18409-18424 (2013).</li><li>Wan, G., Gomez-Casati, M. E., Gigliello, A. R., Liberman, M. C., Corfas, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophin-3+regulates+ribbon+synapse+density+in+the+cochlea+and+induces+synapse+regeneration+after+acoustic+trauma.">Neurotrophin-3 regulates ribbon synapse density in the cochlea and induces synapse regeneration after acoustic trauma.</a> Elife. 3, (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> Journal of Neuroscience. 33 (34), 13686-13694 (2013).</li><li>Furman, A. C., Kujawa, S. G., 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=Noise-induced+cochlear+neuropathy+is+selective+for+fibers+with+low+spontaneous+rates.">Noise-induced cochlear neuropathy is selective for fibers with low spontaneous rates.</a> Journal of Neurophysiology. 110 (3), 577-586 (2013).</li><li>Kujawa, S. G., 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=Adding+insult+to+injury:+cochlear+nerve+degeneration+after+"temporary"+noise-induced+hearing+loss.">Adding insult to injury: cochlear nerve degeneration after "temporary" noise-induced hearing loss.</a> Journal of Neuroscience. 29 (45), 14077-14085 (2009).</li><li>Valero, M. D., 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=Noise-induced+cochlear+synaptopathy+in+rhesus+monkeys+(Macaca+mulatta).">Noise-induced cochlear synaptopathy in rhesus monkeys (Macaca mulatta).</a> Hearing Research. 353, 213-223 (2017).</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>Tong, M., Brugeaud, A., Edge, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerated+synapses+between+postnatal+hair+cells+and+auditory+neurons.">Regenerated synapses between postnatal hair cells and auditory neurons.</a> Journal of the Association for Research in Otolaryngology. 14 (3), 321-329 (2013).</li><li>Landegger, L. D., Dilwali, S., Stankovic, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+murine+cochlear+explant+technique+as+an+in+vitro+screening+tool+in+hearing+research.">Neonatal murine cochlear explant technique as an in vitro screening tool in hearing research.</a> Journal of Visualized Experiments. (124), (2017).</li><li>Takeda, S., Mannstr&#246;m, P., Dash-Wagh, S., Yoshida, T., Ulfendahl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Aging+and+Noise+Exposure+on+Auditory+Brainstem+Responses+and+Number+of+Presynaptic+Ribbons+in+Inner+Hair+Cells+of+C57BL/6J+Mice.">Effects of Aging and Noise Exposure on Auditory Brainstem Responses and Number of Presynaptic Ribbons in Inner Hair Cells of C57BL/6J Mice.</a> Neurophysiology. 49 (5), 316-326 (2017).</li><li>Mehraei, 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=Auditory+brainstem+response+latency+in+noise+as+a+marker+of+cochlear+synaptopathy.">Auditory brainstem response latency in noise as a marker of cochlear synaptopathy.</a> Journal of Neuroscience. 36 (13), 3755-3764 (2016).</li></ol>

由于耳蜗突触病首先在成年小鼠中具有临时阈值偏移(TTS)的特征,由8\u201216 kHz八度频带噪声在100 dB SPL为2 h31,研究人员已经越来越多地研究突触病在各种哺乳动物,包括猴子和人类32,33。除了噪音暴露,其他几个条件与耳蜗性突触病变(例如,老化,使用耳毒性药物和基因突变),导致超阈值试镜的短期中断,其次是不可逆转的听觉神经的退化。在?...

频率在大约 20μu201220,000 Hz 的范围内,人类可视为听觉刺激。人的听力通常最敏感,接近1,000赫兹,其中平均声压水平为20μPa在年轻人(即0分贝的声压水平[dB SPL])。在某些病理条件下,听力损失仅限于特定频率。例如,在噪声引起的听力损失 (NIHL) 的早期阶段,可在 4 kHz1的音频图中观察到"切口"(即听力阈值高程)。沿着哺乳动物耳蜗分区,其刚度和质量的梯度产生一个指数频率图,在耳蜗底部进行高频声音检测,在尖点2进行低频检测。事实上,有一个耳蜗位置频率图沿巴西拉膜,导致所谓的同位素组织2,3。在巴西拉膜上每个给定的位置对只有一个特定的声音频率具有最高的灵敏度,这通常称为特征频率3,4,尽管也可以观察到对其他频率的响应。
迄今为止,各种小鼠模型已被应用于研究听觉系统的正常功能、病理过程和疗效。准确了解小鼠耳蜗的生理参数是听力损失研究的先决条件。小鼠耳蜗在解剖学上分为锥形、中间和基底转弯,对应于不同的频率区域。通过在耳蜗核上标记听觉神经关联,以分析耳蜗中相应的周围内侧位点,Müller等人成功地在体内正常小鼠中建立了耳蜗位置频率图。在 7.2~61.8 kHz 的间隔内,对应于巴西拉膜全长 90% 到 10% 之间的位置,鼠标耳蜗位置频率图可以通过简单的线性回归函数来描述,这表明与耳蜗基的标准化距离和特征频率5的对数。在实验鼠中,位置频率图可用于探索特定频率范围内的听觉阈值与显示沿巴西拉膜6相对区域缺失毛细胞数量的耳蜗图之间的关系。重要的是,位置频率图提供了一个定位系统,用于调查最小的结构损伤,例如毛细胞在周围听觉创伤的特定耳蜗频率位置的带状突触损伤7 ,8.
在哺乳动物耳蜗中,带状突触由先发前带、电子密集投影组成,在IHC内系上含有谷氨酸的释放就绪突触囊泡的光环,以及SGN神经端端的贴片密度谷氨酸受体9。在耳蜗声音转导过程中,发细胞束的偏转导致IHC去极化,导致谷氨酸释放从IHC释放到鼻后发泡端,从而激活听觉通路。此通路的激活导致声音诱导的机械信号在 SGN10中转换为速率代码。事实上,IHC 色带突触高度专业化,能够以数百赫兹的速度以高时间精度进行不知疲倦的声音传输,并且对于声音编码的预突触机制至关重要。先前的研究表明,在成年小鼠耳蜗11、12的不同频率区域,带状突触在大小和数量上差异很大,可能反映对特定声音编码的结构适应。生存需求。最近,实验动物研究表明,耳蜗突触导致多种形式的听力损伤,包括噪音引起的听力损失,年龄相关的听力损失,和遗传性听力损失1314.因此,利用通过基因或环境变量15、16、17的实验操作。
在本报告中,我们提出了一个协议,用于分析成年小鼠巴西拉膜的特定频率区域的突触数、结构和功能。耳蜗频率定位使用给定的位置频率映射与耳蜗图结合执行。通过前突触和午睡免疫染色评估耳蜗带突触的正常形态特征。耳蜗带突触的功能状态是根据ABR波I的超阈值振幅确定的。通过细微的改动,此协议可用于检查其他动物模型(包括大鼠、豚鼠和老年病)的生理或病理状况。

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			<td height="21" style="height:21px;width:216px;">Ketamine hydrochloride</td>
			<td style="width:304px;">Gutian Pharmaceutical Co., Ltd., Fujian, China</td>
			<td style="width:141px;">H35020148</td>
			<td style="width:167px;">100mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine hydrochloride</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>X-1251</td>
			<td>10mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TDT physiology apparatus</td>
			<td>Tucker-Davis Technologies, Alachua, FL, USA</td>
			<td colspan="2">Auditory Physiology System III</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SigGen/BioSig software</td>
			<td>Tucker-Davis Technologies, Alachua, FL, USA</td>
			<td colspan="2">Auditory Physiology System III</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electric Pad</td>
			<td>Pet Fun</td>
			<td>11072931136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont forceps 3#</td>
			<td>Fine Science Tools, North Vancouver, B.C., Canada</td>
			<td>0203-3-PO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont forceps 5#</td>
			<td>Fine Science Tools, North Vancouver, B.C., Canada</td>
			<td>0209-5-PO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereo dissection microscope</td>
			<td>Nikon Corp., Tokyo, Japan</td>
			<td>SMZ1270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat serum</td>
			<td>ZSGB-BIO, Beijing,China</td>
			<td>ZLI-9021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-glutamate receptor 2, extracellular, clone 6C4</td>
			<td>Millipore Corp., Billerica, MA, USA</td>
			<td>MAB397</td>
			<td>mouse&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Purified Mouse Anti-CtBP2</td>
			<td>BD Biosciences, Billerica, MA, USA</td>
			<td>612044</td>
			<td>mouse&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 568 goat anti-mouse IgG1antibody</td>
			<td>Thermo Fisher Scientific Inc., Waltham, MA, USA</td>
			<td>A21124</td>
			<td>goat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 goat anti-mouse IgG2a antibody</td>
			<td>Thermo Fisher Scientific Inc., Waltham, MA, USA</td>
			<td>A21131</td>
			<td>goat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounting medium containing DAPI</td>
			<td>ZSGB-BIO, Beijing,China</td>
			<td>ZLI-9557</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal fluorescent microscopy</td>
			<td>Leica Microsystems, Wetzlar, Germany</td>
			<td>TCS SP8 II</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Image Pro Plus software</td>
			<td>Media Cybernetics, Bethesda, MD, USA</td>
			<td>version 6.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Professional diagnostic pocket otoscope</td>
			<td>Lude Medical Apparatus and Instruments Trade Co., Ltd., Shanghai,China</td>
			<td>HS-OT10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Needle electrode</td>
			<td>Friendship Medical Electronics Co., Ltd., Xi&#39;an,China</td>
			<td>1029</td>
			<td>20 mm, 28 G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Closed-field speaker</td>
			<td>Tucker-Davis Technologies, Alachua, FL, USA</td>
			<td>CF1</td>
			<td></td>
		</tr>
	</tbody>
</table>

morphological and functional evaluation of ribbon synapses at specific frequency regions of the mouse cochlea

耳蜗内毛细胞 (IHC) 通过带状突触将声学信号传输到螺旋型神经神经元 (SGNs)。几项实验研究表明,毛细胞突触可能是感觉神经性听力损失(SNHL)的初始靶点。这些研究提出了耳蜗"突触"的概念,它是指在色带突触数、结构或功能上发生改变,导致IHCs和SGN之间异常突触传播。虽然耳蜗性突触是不可逆的,但它不影响听力阈值。在噪声诱导的实验模型中,对特定频率区域的IHC突触的有限损伤被用来识别特别引起突触性的环境因素,以及干扰内耳的生理后果电路。在这里,我们提出了一个协议,用于分析耳蜗突触形态和功能在成年小鼠的特定频率区域。在此协议中,使用位置频率映射与耳蜗图一起执行特定频率区域的耳蜗定位,然后通过突触评估色带突触的形态特征免疫染色。然后根据听觉脑干响应 (ABR) 波 I 的振幅确定带状突触的功能状态。本报告表明,这种方法可用于加深我们对耳蜗突触功能障碍的发病机制和机制的理解,这可能有助于开发新的治疗干预措施。

在麻醉下对10只C57BL/6J小鼠(8周大)进行了ABR听力测试。AVR在4、8、16、32和48 kHz下使用音调突发刺激引起。通过区分ABR中至少一个清晰的波形,可以直观地检测出每个动物的听觉阈值。所有小鼠都表现出ABR阈值,以响应音调爆裂,范围在25至70分贝SPL之间,取决于刺激的频率。我们的结果表明,听力阈值最低,为16 kHz(图1),相当于与耳蜗顶约43%的距离(图2),表明其他耳...

Watch this Scientific Journal Video about Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea at JoVE.com

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

本手稿描述了用于评估正常小鼠带状突触的形态特征和功能状态的实验协议。本模型也适用于噪声诱发和与年龄相关的耳蜗性突触性受限模型。讨论了以往小鼠研究的相关结果。

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 ...

主要报告是真正强调准确定位罗勒膜的特定频率区域在大多数模型中，其中突触数改变和功能可以分析。使用特定频率区域的耳蜗定位，将频率图与人工图数据结合使用。显微镜提供了对几项研究的洞察。它支持耳蜗神经病变是听力损失、耳鸣和超粘液之前的主要初始事件的概念。在确认对麻醉的八周大成人 C57 黑色 6J 雄性小鼠的手指捏没有反应后。将鼠标放在 37 摄氏度的热垫上，放在电气和隔音室。接下来，在头骨顶点处放置一个20毫米28分针记录电极，皮肤下的深度为3毫米。在异形帕罗蒂德区域的参考电极，在测量耳的针脚下面，在反边帕罗蒂德区域的接地电极。将装有两厘米塑料管的封闭式现场扬声器放在鼠标旁边，将尖端安装到外部耳道中。对于听觉脑干反应（或 ABR 记录），在声压水平从 90 分贝降低至 10 分贝时生成音点，以 5 到 10 分贝的声音压力级别步长。在此步骤中，将放大、筛选和平均响应。在每个频率下，确定 ABR 阈值，即最小声压级别，用于进行可靠的 ABR 录制，并具有一个或多个可区分的波，可以通过目视检查清晰识别。ABR 录音后，从腹侧露出牛皮，用锋利的剪刀打开牛皮，进入耳蜗。使用精细钳子，去除时间骨骼，切断动脉。然后从椭圆形窗口上取下薄片，并破裂圆形窗膜。轻轻旋转 13 毫米 27 表针的尖端，在耳蜗的顶点处打一个小孔，然后使用细尖移液器将 4% 的甲醛通过窗户轻轻冲洗到正食空间。接下来，用冷清新的0.1摩尔PBS每次洗涤，冲洗骨头三次，每次洗涤五分钟。接着是骨骼的脱钙，在室温下用 10%EDTA 进行 4 小时，在水平摇床上每分钟旋转 20 次。在孵化结束时，在解剖显微镜下将一个分切的时骨转移到新鲜的0.1摩尔PBS中。使用 3 和 5 Dumont 钳子和 27 量表针来解剖紫锥、中角和基底耳蜗区域。使用剃须刀刀片沿螺旋韧带进行一小系列切口，并去除气管膜和瑞斯纳的膜。进一步解剖剩余的听觉上皮，包括螺旋肢体，到单独的耳蜗转整个安装准备。然后使用光学显微镜的 40 倍油目标测量罗勒膜长度，将 250 微米刻度放置在眼片中。它可以沿着内毛细胞的立体体进行调整。解剖后，将每个耳蜗变成单独的2.5毫升离心管。在0.1%Triton X-100中，在室温下在旋转器上用PBS中10%山羊血清阻断任何非特异性结合，在室温下阻断一小时。在孵育结束时，使用200微升移液器尖端在解剖显微镜下去除溶液。在旋转器上，在0.1%Triton X-100中，用5%山羊血清和PBS中稀释的原抗体孵育样本。第二天早上，用每次洗涤的0.1摩尔PBS冲洗组织样品三次，五分钟，以去除任何残留的原抗体。然后在旋转器的室温下，在室温下用PBS中5%山羊血清稀释的5%山羊血清中稀释的适当的二次抗体标记标本，在室温下2至3小时。在孵育结束时，用0.1摩尔PBS洗涤样品三次，并转移样品到含有0.1摩尔PBS的单个35毫米板中。将一滴 DAPI 补充安装介质放在幻灯片上，然后将试样从 PBS 转移到安装介质上。将盖滑的一条边放到每个滑轨上并松开，让盖滑轻轻落下。然后在四摄氏度的幻灯片箱中干燥幻灯片。要对标本进行成像，请使用带适当激光和 63X 高分辨率油浸透镜的共合显微镜，从每个人工耳蜗转动获取 8 微米共合 Z 堆栈。对于突触朋塔计数，设置具有 0.3 微米步长的 Z 堆栈以跨越内毛细胞的整个长度，确保可以对所有突触朋塔进行成像并合并 Z 堆栈中包含图像的 puncta 以获取 Z 访问投影。将合并的图像导入适当的图像处理软件程序。将特定频率区域的每个 Z 堆栈中的突触总计计数除以 DAPI 正内毛细胞的数量，以计算每个单元的突触pucta数。在每个特定的频率区域，平均所有突触朋克塔在三个不同微观场的图像，包含9至11个内毛细胞。为了更好地可视化细胞骨骼结构和突触定位，请使用画笔工具直观地评估单个内毛细胞的突触结构和分布。要检查预突触带和后突触受体贴片的并列，请使用矩形选框工具提取色带周围的体素空间，并使用图像剪贴来隔离各个色带。然后单击图像和图像大小，获取这些微型投影的缩略图数组，这些投影可用于识别配对的突触与孤立功能区。这个代表性实验中的所有10只小鼠都表现出 ABR 阈值，以响应每个声压级别 25 到 70 分贝的音调爆发，具体取决于刺激的频率。此实验的听力阈值最低为 16 千赫，对应与耳蜗顶点大约 43% 的距离，这表明其他人工耳蜗区域的声学灵敏度显著降低。听觉上皮的整个坐骑被解剖成三块，长度被测量并转换成它们与耳蜗顶点的距离百分比。使用对数函数计算每个耳蜗转动的罗勒膜上的频率位置。在成年小鼠的正常耳朵中，免疫污染揭示了突触带和谷氨酸受体斑块的并列对。研究内毛细胞的基底膜表面，每个细胞有8到20对。虽然绝大多数的puntta在正常耳朵中显示为并列对，但孤儿丝带在高放大率下很少被观察到。内毛细胞带突触的数量在 16 千赫区域最高，并且随着距离此位置的距离的增加而显著减少。在这个过程中，最重要的是要记住，必须保证人工耳蜗基膜的完整性。我们需要强调准确性，直到达到技术熟练程度。按照这个程序，可以进行基因治疗，以回答耳蜗神经病变是否可以通过这个措施修复。此措施是探索耳蜗神经病变的更广泛的技术，如果主要初始事件与有听力损失，耳鸣和超耳塞有关。

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Neuroscience

Unit 1

The Special Senses

SCIENTISTS IN ACTION

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

与耳蜗内毛细胞传入树突：多泡发布监测功能区突触的突触后录音

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the ...

科研

神经科学

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). ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

在小鼠大脑的各个区域端粒酶活性：非放射性端粒酶重复序列扩增法（TRAP）分析

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

隔膜的神经支配膈运动神经元的功能和形态评估

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

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 ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

全山解剖和成年小鼠耳蜗的免疫

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

小鼠初级少突胶质的快速特异磁分离

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

活的嗅觉途径的功能评价

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

前体小鼠膈肌三方突触细胞特异性钙信号的影像学研究

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns

刺激特定的皮质视觉引发潜在形态模式

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using ...