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신경과학

小鼠耳蜗特定频率区域带状带突起的形态和功能评估

Published: May 10, 2019
doi:
10.3791/59189
, , , , , , , , ,
1Department of Otolaryngology-Head and Neck Surgery, Beijing Friendship Hospital,Capital Medical University, 2Department of Otology,Shengjing Hospital of China Medical University

Summary

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。

Abstract

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。

Introduction

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。

Protocol

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 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?…

Representative Results

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…

Discussion

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?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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)。

Materials

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

Keywords: CochleaFrequency RegionSynapseNeuropathyHearing LossTinnitusHyperacusisAuditory Brainstem ResponseABRCochlea DissectionParaformaldehydeEDTAMicroscopy
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Yu, S., Du, Z., Song, Q., Qu, T., Qi, Y., Xiong, W., He, L., Wei, W., Gong, S., Liu, K. Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea. J. Vis. Exp. (147), e59189, doi:10.3791/59189 (2019).
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