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.
