CELLULAR BASIS OF TUNING IN THE COCHLEA

耳蜗调节的细胞基础

• 批准号: 3216916
• 负责人: JONATHAN JAMES ART
• 金额: $ 12.67万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-02-01 至 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3216916
• 关键词: Chelonia; afferent nerve; biological signal transduction; calcium flux; chemical kinetics; ciliary ganglion; cochlea; cochlear microphonic potentials; confocal scanning microscopy; ear hair cell; efferent nerve; fluorescent dye /probe; microelectrodes; neural conduction; potassium channel; sound frequency; ultraviolet radiation; voltage /patch clamp

The goal of the proposed research is to characterize the cellular mechanisms that contribute to tuning in the turtle cochlea. By understanding how these cellular mechanisms determine frequency selectivity, we will be able to assess their limitations and applicability to hearing in higher vertebrates including man. The proposed research will study the innate mechanical and electrical basis of tuning and modulation by efferent input. A combination of patch clamp, microelectrode, and confocal imaging techniques will be used on isolated, solitary hair cells and in the intact basilar papilla. Previous measurements of ionic currents in solitary cells will be extended and combined with [Ca2+]i imaging to reconstruct membrane potential resonance. Transduction in solitary cells will be analyzed to determine if tuning due to basolateral conductances is enhanced by active, mechanical processes in the ciliary bundle. Finally, efferent modulation of tuning will be investigated in both solitary cells and the intact papilla. Special attention will be paid to the need to unambiguously determine the ionic basis of efferent action and whether the synaptic conductances are identical to those involved in electrical tuning. These results will be used to construct a complete description of the cellular mechanisms involved in tuning. Experiments will determine whether ciliary bundle motion in turtle hair cells is produced by a change in stiffness or a voltage-dependent force. The site and cellular mechanism underlying the motion will be characterized. In further experiments on the transducer, the channels at sites of transduction along the length of the stereocilia and the apical surface of the hair cell will be inactivated with UV radiation. The currents of solitary cells will be measured with the perforated-patch variation of the whole-cell voltage-clamp technique. Changes in the size and kinetics of the membrane currents in cells of known characteristic frequency will be analyzed using intracellular exchange of monovalent cations. Simultaneous cell-attached single-channel and perforated-patch whole cell recordings will be used to compare the behavior of the single channel and the macroscopic IK(Ca). Changes in [Ca2+]i will be measured simultaneously using confocal imaging of indo-1 fluorescence. The single channel will then be studied in an excised patch, and its voltage- and [Ca2+] - sensitivity assessed. Confocal imaging of indo-1 fluorescence will be used to measure the local variation of [Ca2+]i within a single hair cell in the intact papilla. Confocal imaging of electrically activated afferent and efferent fibers stained with the voltage-sensitive dye, di-4-ANEPPS, will be used to identify afferent and efferent terminals on the hair cell's basolateral surface. Hair cell potential will be controlled with a single microelectrode used in current clamp or in switching-mode voltage clamp. The spatial changes of [Ca2+]i in the hair cell that occur when the cell is depolarized, during mechanical stimulation, or that following direct electrical stimulation of the efferent fibers will be compared with the position of afferent and efferent terminals.

拟议研究的目的是表征细胞 有助于在乌龟耳蜗中调整的机制。 经过 了解这些细胞机制如何确定频率 选择性，我们将能够评估他们的局限性和适用性 在包括人在内的更高脊椎动物中听到。 拟议的研究将 研究调整和调制的先天机械和电气基础 通过输入。 斑块夹，微电极和 共聚焦成像技术将用于孤立的，孤立的毛细胞 和完整的基底乳头。 离子电流的先前测量 在孤立的单元中，将扩展并与[Ca2+] i成像合并到 重建膜潜在共振。 孤立细胞的转导 将分析以确定基底外侧电导引起的调整是否为 通过睫状束中的主动机械过程增强。 最后， 在两个孤立细胞中将研究调谐的传出调制 和完整的乳头。 特别注意需要 明确确定传出作用的离子基础，以及是否是否 突触电导与参与电气调谐的电导相同。 这些结果将用于构建对 调音涉及的细胞机制。 实验将确定乌龟头发中的睫状束运动是否 细胞是由刚度变化或电压依赖力产生的。 运动基础的位点和蜂窝机理将是 特征。 在对传感器的进一步实验中，通道处 沿着立体胶质和顶端的长度转导的位点 毛细胞的表面将被紫外线辐射灭活。 孤立单元的电流将用穿孔点进行测量 全细胞电压钳技术的变化。 大小的变化 和已知特征细胞中膜电流的动力学 将使用单价的细胞内交换来分析频率 阳离子。 同时与细胞连接的单通道和穿孔整个细胞 录音将用于比较单个通道的行为和 宏观IK（CA）。 [Ca2+]的更改将同时测量 使用Indo-1荧光的共聚焦成像。 单个通道将 然后在切除的贴片中研究，其电压和[Ca2+] - 敏感性评估。 Indo-1荧光的共聚焦成像将用于测量局部 完整乳头中的单个毛细胞内[Ca2+] i的变化。 电活化传入和泡沫纤维的共聚焦成像 用对电压敏感的染料染色DI-4-ANEPPS将用于 识别毛细胞基底外侧的传入和传出终端 表面。 毛细胞电位将通过一个单一控制 当前夹具或开关模式电压夹中使用的微电极。 在电池为当时发生的毛单元中[Ca2+] i的空间变化 在机械刺激期间或直接刺激期间去极化 将将传出纤维的电刺激与 传入和传出终端的位置。