The receptor current of hair cells from the bullfrog's sacculus was measured by voltage clamp recording across the isolated sensory epithelium. Several hundred hair cells were stimulated en masse by moving the overlying otolithic membrane with a piezoelectrically activated probe. As measured by optical recording of otolithic membrane motion, the step displacement stimuli reached their final amplitudes of up to 1 micrometer within 100 microseconds. The relationship between displacement and steady-state receptor current is an asymmetric, sigmoidal curve about 0.5 micrometer in extent. The time constant of the approach to steady state depends upon the magnitude of the hair bundle displacement and ranges from 100 to 500 microseconds at 4 degrees C; the time course is faster with larger displacements or at higher temperatures. Both the displacement-response curve and the kinetics of the response are changed by alterations in the Ca2+ concentration at the apical surface of the cells. The characteristics of the response are not consistent with simple models for the transduction process that involve enzymatic regulation of channel proteins or diffusible second messengers. Mechanical stimulation is instead posited to act directly by altering the free energy difference between the open and closed forms of the transduction channel, thereby inducing a redistribution between these states. The dependences of the response kinetics on displacement and on temperature suggest that the thermal interconversion between open and closed transduction channels is limited by an enthalpy of activation of about 12 kcal/mol.

Hair cells, the primary receptors of the auditory, vestibular, and lateral-line sensory systems, produce electrical signals in response to mechanical stimulation of their apical hair bundles. We employed an in vitro preparation and intracellular recording to investigate the transduction mechanism of hair cells in the sacculus from the inner ear of the bullfrog (Rana catesbeiana). When Hair cells respond with small receptor potentials (1, 2) which evidently excite afferent nerve fibers by chemical (3) or electrical synapses.The transduction process by which bending of the hair bundle elicits an electrical response is poorly understood because hair cells are generally small, relatively inaccessible, and difficult to impale with intracellular microelectrodes. Moreover, because the cells in situ are stimulated through the mechanical and hydrodynamic linkages that couple their hair bundles to vibrations in the external medium, the exact nature of the mechanical stimuli delivered to the cells is unknown in every instance. We have circumvented these difficulties by developing an in vitro preparation which permits intracellular recording from relatively large hair cells during the delivery of precisely defined stimuli directly to their hair bundles.MATERIALS AND METHODS Experimental Preparation. The hair cell preparation was taken from the sacculus of the bullfrog (Rana catesbeiana); this organ of the inner ear responds to ground-borne vibrations (4) and perhaps to sound (5). The macula, a discoidal region of the saccular wall in which the hair cells are situated (6), was rapidly dissected from the labyrinth by a ventral surgical approach. After the otoconia (otoliths) within the sacculus were rinsed away with a gentle stream of standard saline solution, the transparent otolithic membrane that overlies the macula was carefully peeled away with fine forceps.

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Adaptation in a vestibular organ, the bullfrog's sacculus, was studied in vivo and in vitro. In the in vivo experiments, the discharge of primary saccular neurons and the extracellular response of saccular hair cells were recorded during steps of linear acceleration. The saccular neurons responded at the onset of the acceleration steps, then adapted fully within 10-50 msec. The extracellular (microphonic) response of the hair cells adapted with a similar time course, indicating that the primary sources of the neural adaptation are peripheral to the afferent synapse--in the hair cell, its mechanical inputs, or both. Evidence for hair cell adaptation was provided by 2 in vitro preparations: after excising the sacculus and removing the accessory structures, we recorded either the extracellular hair cell response to displacement of the otolithic membrane or the intracellular hair cell response to hair bundle displacement. In both cases the response to a step stimulus adapted. The adaptation involved a shift in the displacement-response curve along the displacement axis, so that the cell's operating point was reset toward the static position of its hair bundle. This displacement shift occurred in response to both depolarizing and hyperpolarizing stimuli. Its time course varied among cells, from tens to hundreds of milliseconds, and also varied with the concentration of Ca2+ bathing the apical surfaces of the hair cells. Voltage-clamp experiments suggested that the displacement shift does not depend simply on ion entry through the hair cell's transduction channels and can occur at a fixed membrane potential. The possible role of the displacement-shift process in the function of the frog's sacculus as a very sensitive vibration detector is discussed.

Bullfrog saccular hair cells adapt to maintained displacements of their stereociliary bundles by shifting their sensitive range, suggesting an adjustment in the tension felt by the transduction channels. It has been suggested that steady-state tension is regulated by the balance of two calcium-sensitive processes: passive "slipping" and active "tensioning." Here we propose a mathematical model for an adaptation motor that regulates tension, and describe some quantitative tests of the model. Slipping and tensioning rates were determined at membrane potentials of -80 and +80 mV. With these, the model predicts that the I(X) curve (relating bundle displacement and channel open probability) should shift negatively by 124 nm when the cell is depolarized, with an exponential time course that is slower on depolarization from -80 to +80 mV than on repolarization. This was observed: on depolarization, the I(X) curve shifted by an average of 139 nm, and displayed the expected difference in rates at the two potentials. Because the negative shift of the I(X) curve on depolarization represents an increase in the tension on transduction channels, the model also predicts this tension should cause an unrestrained bundle to pivot negatively by 99 nm on depolarization. Such movement was observed using high-resolution video microscopy; its amplitude was variable but ranged up to about 100 nm, and its time course was asymmetric in the same way as that of the I(X) curve shift. In additional comparisons, the active bundle movements and I(X) curve shift exhibited a similar steady-state voltage dependence, and were both reversibly abolished by reduced bath Ca2+ or by the transduction channel blocker streptomycin. Lastly, among different cells, the amplitude of the movement increased with the size of the transduction current. Thus, a quantitative mechanical model for adaptation also accounts for the observed mechanical behavior of the bundle, suggesting that the same mechanism is responsible for both, and that adaptation is mediated by an active, force-producing mechanism.