产后大鼠听觉脑干反应及外毛细胞全细胞贴片钳记录
Summary
本协议描述的方法, 记录的听觉脑干反应的产后鼠幼崽。为研究外发细胞的功能发育情况, 本文分步介绍了全细胞膜片钳在分离外毛细胞中的实验过程。
Abstract
外毛细胞是哺乳动物耳蜗内两种感觉毛细胞之一。它们通过受体电位改变细胞长度, 放大低电平声信号的弱振动。OHCs 外毛细胞的形态和电生理特性在产后早期发育。外毛细胞的成熟可能有助于听觉系统的发展。然而, OHCs 的发展过程还没有得到很好的研究。这部分是由于难以用电生理方法来测量它们的功能。为了开发一种简单的方法来解决上述问题, 我们在这里描述了一步一步的协议, 以研究 OHCs 在急性离型耳蜗从产后大鼠的功能。通过这种方法, 我们可以对纯色调刺激的耳蜗反应进行评价, 并对 OHCs 中运动蛋白 prestin 的表达水平和功能进行研究。该方法也可用于研究内毛细胞 (IHCs)。
Introduction
耳蜗感觉毛细胞的两个不同功能是哺乳动物听觉的关键: mechanoelectric 转导 (见) 和 electromotility1,2。通过在发束中遇到的通道, IHCs (IHCs) 和 OHCs 将声音振动转换成膜电位的变化, 以及支配螺旋神经节神经元的电信号。OHCs 改变其细胞长度与膜电位, 放大低电平声音的振动。这种活动称为 electromotility 是由位于 OHCs3侧壁的运动蛋白 prestin 所衍生。
在许多物种中, 包括啮齿类动物, 听觉功能是不成熟的早期出生的时代4,5。在听觉皮层中, 在听觉皮质上没有任何动作电位可以检测到声音信号6,7。耳蜗的形态学和功能的发展已被广泛研究的老鼠, 沙鼠, 鼠4,5,8。毛发细胞的 mechanotransduction 和 electromotility 也在早期生命世纪的5中发展起来。
为评价大鼠不同出生年龄的听觉敏感性, 研制了一种听觉脑干反应 (ABR) 记录方法。全细胞贴片钳是研究 OHCs electrophysiologically 的理想技术。然而, 与神经元和其他上皮细胞中的膜片钳相比, 全细胞封闭率低, 限制了孤立 OHCs electromotility 的调查。
在这里, 我们描述了一个程序, 以调查 OHCs 的形态和 electrophysiologically 在急性分离的耳蜗从产后大鼠。该方法可对调节内毛细胞发育和功能的分子机制进行改进。
Protocol
Representative Results
Discussion
在11岁以下的老鼠中, 听觉皮层可能没有反应出对声音刺激的动作电位6,7。因此, 后天11被描述为 “听觉发作”10。听力发病前听力功能的发展尚未得到很好的研究。使用类似的方法, 成人 ABR 记录, 我们证明, ABRs 可以引起的纯音调爆裂的鼠幼崽比 P11 (图 1)。然而, 从大鼠幼崽获得的 ABR 波形显示出与成年动物不同的特?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了973计划 (2014CB943002) 和中国国家自然科学基金 (11534013, 31500841) 赠款的支持。
Materials
| Anesthetic | |||
| Pentobarbital sodium | Sigma | P3761 | 1.5% in water |
| Name | Company | Catalog Number | Comments |
| Dissection solution | |||
| Leiboviz's L-15 Medium | Life Technologies | 41300-039 | 1 pack in 1 L water |
| Collagenase IV | Sigma | C5138 | 2 mg/mL in L-15 |
| HEPES | Sigma | 7365-45-9 | 10 mM |
| Name | Company | Catalog Number | Comments |
| Immunostaining solutions | |||
| PBS | Thermo Fisher Scientific | 10010023 | PH 7.3 |
| Paraformaldehyde | Sigma | 158127 | 4% in PBS |
| Triton X-100 | Amresco | ZS-0694 | 0.3% in PBS |
| Normal goat serum | Thermo Fisher Scientific | 10000C | 10% in PBS |
| prestin antibody | Santa Cruz | SC-22694 | dil 1:200 |
| Alexa Fluor 488-conjugated antibody | Thermo Fisher Scientific | A-11055 | dil 1:600 |
| Phalloidin-Tetramethylrhodamine B isothiocyanate | Sigma | P1951 | dil 1:200 |
| DAPI | Solarbio | C0060 | dil 1:20 |
| Name | Company | Catalog Number | Comments |
| Extracellular solution | |||
| Leiboviz's L-15 Medium | Life Technologies | 41300-039 | 1 pack in 1 L water |
| HEPES | Sigma | 7365-45-9 | 10 mM |
| Name | Company | Catalog Number | Comments |
| Intracellular solution | |||
| CsCl | Sigma | 7647-17-8 | 140 mM |
| MgCl2 | Sigma | 7791-18-6 | 2 mM |
| EGTA | Sigma | 67-42-5 | 10 mM |
| HEPES | Sigma | 7365-45-9 | 10 mM |
| Name | Company | Catalog Number | Comments |
| Equipment | |||
| Osmometer | Gonotec | OSMOMAT 3000 basic | |
| Forcep | WPI | 14095 | Tweezers dumont |
| Micropipette puller | Sutter Instrument | MODLE-P97 | |
| Micro Forge | Narishigen | MF-830 | |
| Mini Operating System | Sutter Instrument | MP-285 | |
| MultiClamp | Axon | 700B | |
| Low-Noise Data Acquisition System | Axon | 1440A | |
| ES1 speaker | Tucker-Davis Technologies | ||
| TDT system 3 | Tucker-Davis Technologies | ||
| Name | Company | Catalog Number | Comments |
| Software | |||
| SigGenRP software | Tucker-Davis Technologies | ||
| BioSigRP software | Tucker-Davis Technologies | ||
| jClamp | Scientific Solutions | ||
| Name | Company | Catalog Number | Comments |
| Animal | |||
| SD rat | Experimental Animal Center of Southern Medical University |
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