Özet

脑干中的数据采集与分析 在小鼠中引发响应音频测量

Published: May 10, 2019
doi:

Özet

脑干引起反应听力测定是临床神经生理学的重要工具。如今,脑干引起反应听力学也应用于基础科学和临床前研究,涉及药理学和遗传动物模型。在这里,我们提供了如何成功地记录和分析小鼠的听觉脑干反应的详细描述。

Abstract

脑干引起反应听力测量(BERA)在临床神经生理学中具有中心相关性。由于其他唤起电位 (EP) 技术,如视觉唤起电位 (VE) 或躯体感官唤起电位 (SEP),听觉唤起电位 (AEP) 由重复呈现相同的刺激触发,脑电图 (EEG) 反应随后被平均,导致明显的正 (p) 和负 (n) 偏转。在人类中,单个峰的振幅和延迟都可以用来描述底层神经元电路中同步和传导速度的变化。重要的是,AEP还应用于基础和临床前科学,以识别和表征药理学和遗传动物模型中的听觉功能。此外,还利用动物模型与药理学测试相结合,研究在治疗感觉神经性听力损失(例如,年龄或噪音引起的听力缺陷)方面的潜在益处。在这里,我们提供了如何使用点击和音调爆裂应用程序记录小鼠的听觉脑干反应 (ADR) 的详细和综合描述。该协议的一个具体重点是实验前动物外壳、麻醉、ABR记录、ABR过滤过程、基于小波的自动振幅生长功能分析和延迟检测。

Introduction

大脑生理学的一个中心方面是处理环境信息的能力,从而产生不同的内在或外在输出,如学习、记忆、情绪反应或运动反应。各种实验和诊断方法可用于描述与刺激相关的神经元回路中单个神经元细胞类型或神经元簇/集合的电生理反应。这些电生理技术覆盖了微尺度、中尺度和宏观尺度上不同的时空尺寸。微尺度电平包括不同贴片夹模式的电压和电流钳位方法,例如,使用培养或急性分离神经元1。这些体外技术允许表征单个当前实体及其药理调制2,3。然而,一个基本缺点是缺乏关于微和宏观电路信息集成和处理的系统信息。这种损伤部分克服了体外技术中尺度,如多电极阵列,允许同时细胞外多电极记录不仅在培养神经元,但也在急性脑切片4,5.微电路可以在特定程度(例如,在海马区)中保存在脑切片中,而远距离互连通常损失6。最终,为了研究神经元电路中的功能互连,在宏观上进行系统体内电生理技术是首选方法7。这些方法包括,除其他外,表面(硬膜外)和深(脑内)脑电脑记录,在人类和动物模型1中进行。脑电图信号主要基于不同皮质层中金字塔神经元的同步突触输入,尽管兴奋输入具有普遍优势,但在主体中可以抑制或兴奋。同步时,在细胞外电场中,激发性后视位电位被求和,形成足够强度的信号,使用表面电极记录在头皮上。值得注意的是,从单个电极检测的头皮记录需要一万个金字塔神经元的活动,以及技术设备和加工工具的复杂武器库,包括放大器、过滤过程(低通滤波器、具有特定导体特性的高通滤波器、陷波滤波器和电极。

在大多数实验动物物种(即小鼠和大鼠)中,基于人的头皮 EEG 方法在技术上不适用,因为由于同步金字塔神经元的数量有限,底层皮层产生的信号太弱, 10,11.因此,在啮齿类动物中,表面(头皮)电极或皮下电极受到心电图和主要电图伪影的严重污染,使得高质量的脑电图记录不可能9、1112.因此,当使用未经麻醉的自由移动小鼠和大鼠时,必须通过硬膜外电极或从深层的脑内结构直接从皮层进行记录,以确保传感尖端的直接物理连接铅/植入电极到信号生成神经元细胞簇。这些脑电图方法可以在约束系绳系统设置中进行,也可以使用非约束植入式EEG无线电遥测方法9、10、11。这两种技术都有其优缺点,在癫痫发作易感性/癫痫活动、昼夜节律、睡眠结构、振荡活动和同步的定性和定量表征中,都是有价值的方法,包括时间频率分析、源分析等9、10、13、14、15、16、17。

虽然系绳系统和无线电遥测允许分别在限制/半约束或非约束条件下进行 EEG 记录,但相关的实验条件不符合 ABR 录像的要求。后者对定义的声学刺激的需求,随着时间的推移,通过扬声器和实验动物的固定位置和受控声压水平 (SPL) 重复呈现。这可以通过在限制条件下的头部固定或麻醉后18,19来实现。为了减轻实验压力,动物通常在ABR实验中进行麻醉,但应考虑麻醉会干扰ADR19,20。

作为一般特征,EEG在50-100 μV的电压范围内由不同频率组成。背景频率和振幅在很大程度上取决于实验动物的生理状态。在醒状态下,以低振幅的β(β)和伽马(+)频率占主导地位。当动物昏昏欲睡或入睡时,α (α)、ta (+) 和增量 (+) 频率出现,显示 EEG 振幅增加 21。一旦感觉通道(例如,声学通路)受到刺激,信息传播通过周围和中枢神经系统的神经元活动进行中介。这种感官(例如,声学)刺激触发所谓的EP或引起反应。值得注意的是,与事件相关的电位 (ERP) 的振幅远低于 EEG(即仅几微伏)。因此,任何基于单个刺激的单个 ERP 都会在高振幅 EEG 背景下丢失。因此,ERP 的录制需要重复应用相同的刺激(例如,ABR 录音中的咔嗒声)和随后的平均值,以消除任何 EEG 后台活动和伪影。如果ABR记录是在麻醉动物中完成的,则在这里很容易使用皮下电极。

主要包括短延迟 EP,通常与 ADR 或 BERA 相关,以及进一步、后期启动的电位,如中延迟 IP(中延迟响应 [MLR])和长期延迟 EP22。重要的是,听觉信息信息处理中的干扰通常是神经精神病(脱骨髓疾病、精神分裂症等)的核心特征,并且与AEP改变23、24有关 ,25.而行为调查只能揭示功能障碍,AEP研究允许对与特定神经解剖学结构相关的听觉功能障碍进行精确的时空分析26。

ABR 早期、短延迟声学 EP 通常在中度到高强度的点击应用中检测到,并且可能发生多达七个 ABR 峰值 (WI-WVII)。最重要的波(W I-WV)与以下神经解剖学结构有关:WI与听觉神经(远端部分,内耳内);WII到耳蜗核(听觉神经的近部,脑干终止);WIII到上级寡物复合物 (SOC);WIV到侧侧耳鸣 (LL);WV端接在侧侧下侧 (IC) 内的侧侧乳管 (LL) 的端接(LL ) (补充图 1)。应该指出,W II-WV可能具有多个解剖结构的上升听觉通路有助于他们。值得注意的是,听觉区域的峰值和基础结构的确切相关性尚未完全澄清。

在听力学中,ADR可用作筛查和诊断工具,以及用于外科监测28,29。它对于识别缺损、子宫内膜炎和厌食症(例如,与年龄相关的听力损失、噪音引起的听力损失、代谢和先天性听力损失,以及畸形引起的不对称听力损失和听力缺陷)至关重要。畸形、损伤和肿瘤)28。ADR 也作为对过度活跃、智力受损的儿童或其他无法对常规听力测量作出反应的儿童(例如,在神经/精神疾病(如多动症、MS、自闭症等)的筛查测试。,30)和在开发和手术安装人工耳蜗28。最后,ADR可以提供宝贵的见解,了解神经精神药物的潜在otois副作用,如抗癫痫药31,32。

从药理学或转基因小鼠模型获得的神经生理学知识翻译给人类的价值在许多环境中得到了证明,特别是在小鼠和大鼠听觉范式中的ERPs水平33。 34,35.因此,对小鼠和大鼠听觉信息处理中改变的早期AEP和相关变化的新见解可以转化为人类,在听觉、神经和听觉的表征和内切分方面具有核心重要性。未来的神经精神病。在这里,我们详细介绍了如何在小鼠中成功记录和分析ADR,以用于基本科学、毒理学和药理学目的。

Protocol

所有动物程序都按照德国动物护理理事会的准则进行,所有协议都由当地机构和国家动物护理委员会批准(州乌姆韦尔特,恩维尔劳彻舒茨北莱茵-威斯特法伦州自然、环境和消费部办公室(德国LANUV NRW)。)。作者进一步证明,所有动物实验都是根据国家卫生研究院《实验室动物护理和使用指南》(NIH出版物第80-23号)修订的1996年或《英国动物(科学程序)法》进行的。1986年及相关准则,或欧洲共同体理事会指?…

Representative Results

点击和音调突发引起的ABR记录可用于评估听力阈值差异、振幅增长函数和延迟比较。图 1中描述了 SPL 增加模式下的点击调用 ADR,用于控制装置和两条示例性突变鼠标线,这些线路在 Cav3.2 T 型电压门控 Ca2+通道(即 Cav3.2+/-和 Ca) 中存在缺陷v3.2 空突变体 [Cav3.2-/-])。如上所述,由于人类54、55和</sup…

Discussion

该协议提供了关于如何记录小鼠听觉引起脑干反应的详细和综合描述。它特别侧重于动物预处理、麻醉和潜在的方法混淆因素。后者包括性别、小鼠线、年龄和住房条件。需要注意的是,所有这些因素都会对感觉神经性听力损失和听觉信息处理的基本方面产生影响。因此,听觉特征分析研究的适当分层是强制性的。

AEP 录音的仪器在过去 50-60 年中发生了巨大的变化,如今,商用 ABR …

Açıklamalar

The authors have nothing to disclose.

Acknowledgements

作者要感谢克里斯蒂娜·科尔布博士(德国神经退行性疾病中心[DZNE])和罗伯特·斯塔克博士(DZNE)在动物育种和动物保健方面给予的帮助。这项工作得到了联邦药品和医疗器械研究所的财政支持。

Materials

AEP/OAE Software for RZ6 (BioSigRZ software) Tucker-Davis Technologies (TDT) BioSigRZ
Binocular surgical magnification microscope Zeiss Stemi 2000 0000001003877, 4355400000000, 0000001063306, 4170530000000, 4170959255000, 4551820000000, 4170959040000, 4170959050000
Cages (Macrolon) Techniplast 1264C, 1290D
Carprox vet, 50mg/ml Virbac Tierarzneimittel GmbH PZN 11149509
Cold light source Schott KL2500 LCD 9.705 202
Cotton tip applicators (sterile) Carl Roth EH12.1
Custom made meshed metal Faraday cage (stainless steel, 2 mm thickness, 1 cm mesh size) custom made custom made
5% Dexpanthenole (Bepanthen eye and nose creme) Bayer Vital GmbH PZN: 01578681
Disposable Subdermal stainless steel Needle
electrodes, 27GA, 12mm
Rochester Electro-Medical, Inc. S03366-18
Surgical drape sheets (sterile) Hartmann PZN 0366787
Ethanol, 70% Carl Roth 9065.5
1/4'' Free Field Measure Calibration Mic Kit Tucker-Davis Technologies (TDT) PCB-378C0
Gloves (sterile) Unigloves 1570
Graefe Forceps-curved, serrated FST 11052-10
GraphPad Prism 6 Software, V6.07 GraphPad Prism Software, Inc. https://www.graphpad.com/
Heat-based surgical instrument sterilizer FST 18000-50
Homeothermic
heating blanked
ThermoLux 461265 / -67
Ketanest S (Ketamine), 25mg/ml Pfizer PZN 08707288
Ringer’s solution (sterile) B.Braun PZN 01471434
Matlab software MathWorks, Inc. https://de.mathworks.com/products/matlab.html
Medusa 4-Channel Low Imped. Headstage Tucker-Davis Technologies (TDT) RA4LI
Medusa 4-Channel Pre-Amp/Digitizer Tucker-Davis Technologies (TDT) RA4PA
Microphone PCB Pieztronics 378C01
Multi Field Speaker- Stereo Tucker-Davis Technologies (TDT) MF1-S
Oscilloscope Tektronix DPO3012
Optical PC1 express card for Optibit Interface) Tucker-Davis Systems (TDT) PO5e
Askina Braucel pads (cellulose absorbet pads) B.Braun PZN 8473637
Preamplifier PCB Pieztronics 480C02
RZ6 Multi I/O Processor system (BioSigRZ) Tucker-Davis Technologies (TDT) RZ6-A-PI
0.9% saline (NaCl, sterile) B.Braun PZN:8609255
SigGenRZ software Tucker-Davis Technologies (TDT) https://www.tdt.com/
Software R (version 3.2.1) + Reshape 2 (Version 1.4.1) + ggplot 2 (version 1.0.1) + datatable (version 1.9.4), + gdata (version 2.13.3), + pastecs (version 1.3.18), + waveslim (version 1.7.5), + MassSpecWavelet (version 1.30.0) The R Foundation, R Core Team 2015 Open Source Software (freely distributable)
Sound attenuating cubicle Med Associates Inc. ENV-018V
Standard Pattern Forceps, 12cm and 14.5 cm length FST 11000-12, 11000-14
Leukosilk tape BSN medical GmbH & Co. KG PZN 00397109
Tissue Forceps- 1×2 Teeth 12 cm FST 11021-12
Uniprotect ventilated cabinet Bioscape THF3378
Ventilated cabinet Tecniplast 9AV125P
Xylazine (Rompun), 2% Bayer Vital GmbH PZN 1320422

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Lundt, A., Soos, J., Henseler, C., Arshaad, M. I., Müller, R., Ehninger, D., Hescheler, J., Sachinidis, A., Broich, K., Wormuth, C., Papazoglou, A., Weiergräber, M. Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice. J. Vis. Exp. (147), e59200, doi:10.3791/59200 (2019).

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