CELLULAR BASIS OF TUNING IN THE COCHLEA

拟议研究的目标是表征细胞 有助于海龟耳蜗调谐的机制。 通过 了解这些细胞机制如何决定频率 选择性，我们将能够评估其局限性和适用性 包括人类在内的高等脊椎动物的听力。拟议的研究将 研究调谐和调制的内在机械和电气基础 通过传出输入。 膜片钳、微电极和 共聚焦成像技术将用于分离的孤立毛细胞 以及完整的基底乳头 先前的离子电流测量 在孤立细胞中将被扩展并与[Ca 2 +]i成像相结合， 重建膜电位共振。 孤立细胞中的转导 将进行分析，以确定是否由于基底侧电导调谐， 通过睫状束中的主动机械过程来增强。 最后， 调谐的传出调制将在两个孤立细胞中进行研究 和完整的乳头 将特别注意需要 明确确定传出作用的离子基础，以及 突触电导与电调谐中所涉及的电导相同。 这些结果将被用来构建一个完整的描述 细胞机制参与调谐。 实验将确定乌龟毛中的纤毛束运动是否 细胞是由刚度变化或电压依赖力产生的。 运动的部位和细胞机制将是 表征了 在对换能器的进一步实验中， 沿着静纤毛和顶纤毛长度的转导位点 毛细胞的表面将被紫外线辐射灭活。 用穿孔贴片测量单个细胞的电流 全细胞电压钳技术的变化。 规模变动 和已知特征的细胞中的膜电流的动力学 频率将使用单价的细胞内交换来分析。 阳离子。 同时细胞贴附单通道和穿孔贴片全细胞 记录将用于比较单通道的行为， 宏观IK（Ca）。 将同时测量[Ca 2 +]i的变化 使用indo-1荧光的共焦成像。 单通道将 然后在离体贴片中进行研究，其电压和[Ca 2 +] - 敏感性评估。 indo-1荧光的共焦成像将用于测量局部 完整乳头内单个毛细胞内[Ca 2 +]i的变化。 电激活传入和传出纤维的共聚焦成像 用电压敏感染料di-4-ANEPPS染色，将用于 识别毛细胞基底外侧的传入和传出终末 面 毛细胞电位将由一个单一的 用于电流钳或开关电压钳的微电极。 当毛细胞被破坏时，[Ca 2 +]i在毛细胞中的空间变化 去极化，在机械刺激过程中，或以下直接 传出纤维的电刺激将与 传入和传出终末的位置。

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.