Transduction and tuning by vertebrate hair cells

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Transduction and tuning by vertebrate hair cells A. J. Hudspeth The internal ear, or labyrinth, displays extraordinary sensitivity: an acoustical signal at the human auditory threshold or a just-perceptible acceleration imparts to the ear less than an attowatt o f power, the power presented to an eye by the absorption o f one photon per second. The detectors for such miniscule stimuli are hair cells, specialized mechanoreceptors that transduce inputs into membrane-potential changes. In addition to endowing animals with great sensitivity to sounds, vibrations and accelerations, the organs o f the internal ear are selectively responsive to inputs o f behavioral importance to each species. The hair cells o f the vestibular apparatus, for example, are sensitive to accelerations o f particular orientations, while the receptors in the auditory system characteristically respond best to sounds o f certain frequencies. Hair cells are accordingly not passive transducers; through their electrical and mechanical properties they also contribute to the selection o f stimuli. I discuss here the current evidence bearing on both these roles o f the vertebrate hair cell, that o f transduction and that o f filtering stimuli.

the cross-bridges among nficrofilaanents and the attachments of the filaments to the enveloping membrane. Unfortunately, we thus far have antibodies tha~ enable us to identify in stereocilia only lhosc proteins previously purified from conventional microvilli or other organeUes. If there is a unique constituent of stereocilia that mediates transduction - the inevitable 'hearm' or 'listenin' - we await a ligand with which to label it specifically. Since no ototoxin has been found whose affinity rivals those of the natural neurotoxins, monoclonalantibody technology presently seems the most promising avenue tbr identifying and subsequently purifying the transduction molecule. The hair bundle is morphologically polarized, for the kinocitium always occurs at one edge of the bundle and, whether the kinocilium persists or regresses, the sterocilia always grow longer at that edge than at the opposite. The electrical responsiveness of the hair cell parallels this anatomical asymmetrylS: the cell depolarizes when the hair bundle's tip is deflected towards the edge at which the kinocilium

Structure The mechanoreceptive organelle of the hair cell, the hair bundle, is a tuft of elongate processes protruding from the cell's apical surface (Fig. 1 ). A stimulus reaching the cell displaces the hair bundle's distal tip parallel with the cellular surface, causing each of the constituent processes to pivot at its base. By a mechanism that remains unknown, this motion alters the cell's membrane conductance to cations 1,2°, modulates the ionic current across the membrane, and thus produces a receptor potential. The components of the hair bundle are in general a single true cilium, the kinocilium, and up to about 300 cylindrical stereocilia. The kinocilium is often the site at which naechanical stimuli impinge upon the hair bundle and it may prove important in the bundle's development. However, because the kinocilium regresses in some organs and may he dissected in others without affecting transduction TM, it is thought that the structures responsible for transduction are the stereocilia. In their structural pattern, the stereocilia are essentially outsized microviUi: each consists of a fdamentous core surrounded by a tube of plasma membrane. The predominant structural components of the stereociliary core are actin microfdaments 9. In at least some species, these filaments are extensively cross-linked such that their helical periodicities are in register across the diameter of each stereocilium 7. Associated with the actin are other proteins9 which presumably form such structural elements as

Fig. 1. Scanning electron micrograph o f the apical surface o f a hair cell from the bullfrog's saccalus. The hair bundle includes a single kinocil'uan with a bulbous tip (Kc) and about fifty stereocitia (Sc). Note that the longest stereocilia occur adjacent to the kinocilium and that more distant ones become progressively shorter. The prominent white arrow lies in the cell's plane o f bilateral symmetry and points in the positive dir• ection; m o vement o f t h e hair bundle's tip in this direction depolarizes the cell ti •2a. AroUnd the hair cell lie several supporting cells whose surfaces are studded with microvilli. ( × 16 000)

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stands, while it hyperpolarizes for stimulation in the opposite direction TM. Orthogonal stimuli evoke no response s.23

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Displacement-response relationship A convenient display of the hair cell's mechanical sensitivity is a displacement-response curve (Fig. 2) relating some aspect of the cellular response (conductance change, ionic current, or receptor potential) to a measure of the stimulus (sound pressure or hair-bundle deflection). This relationship has been determined for individual hair cells in the bullfrog's sacculus by direct manipulation of hair bundles 2.~2~3, and in the guinea-pig's cochlea2~ and the turtle's basilar papilla6 by acoustical stimulation. Similar results have been obtained from extracellular recordings of cell populations in the auditory and lateralline organs of fishs'*°. In each instance, the curve is an asymmetrical sigmoid. When the hair bundle is in its resting position, the conductance of the transduction channels is approximately one-fifth of its maximal value and some current flows inwards through these channels. Motion of the hair bundle's tip in the positive direction, towards the edge of the hair bundle where the kinocilium stands, elicits a membrane conductance increase, an augmented inward current, and a depolarization. Stimuli of the opposite sign produce a conductance decrease, diminish the inward current, and result in hyperpolarizations that are several-fold smaller in magnitude than the corresponding depolarizations. There is an asymmetry also in the asymptotic behavior of the displacement-response curve for the two polarities of stimulation. The curve has a relatively sharp inflection and seems to saturate abruptly for negative stimuli. Positive deflections, on the other hand, produce responses that only gradually approach a limiting value. A striking feature of the displacement-response relationship is its narrowness: half the total response occurs over a displacement of only 200 nm at the tips of hair bundles in the bullfrog's sacculus 2. This narrow range of sensitivity poses a significant experimental difficulty, since displacements of this size are difficult to control and to measure. It also points out one of the most interesting aspects of hair cells, that the transduction event corresponds to movements of molecular dimensions =4. Consider, for example, the motions of some components in the bullfrog's saccular hair cell. If a stereocilium indeed moves as a stiff rod pivoting near its base, a half-saturating stimulus deflects it through an angle of only ± l °. At the sites where the tips of adjacent stereocilia come into con-

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Fig. 2. The displacement--response relationships o f two types o f hair cells - those o f the ballfrog's sacculus (0) and o f the red-eared turtle's basilar papilla ( &). The former data are normalized receptor currents in response to hair-bundle displacements produced by a piezoelectric micromanipulator z. The data from the turtle 6are normalized estimates o f transducer conductance plotted against an abscL~sabased on the assumption that a sound-pressure level o f 120 dB leads to deflection o f hair bundle tips by +_1 pro. Both sets o/ result~ show the asymmetrical, sigmoidal form characteristic o f the displacement-response relationship. The continuous curve is generated on the basis o f a model for the transduction process in the frog's hair cells ~.

tact, the same input causes a shear between their apposed membranes in the order of ± 10 nm. If the actin filaments within each stereocilium are free to slide past one another, the stimulus displaces each by less than ± 200 pm with respect to its neighbors. These minute changes occur with behaviorally very large stimuli; at the effective threshold displacement for transduction, which probably lies near ± 200 pm for these cells, the corresponding motions are smaller by about three orders of magnitude. The asymmetry of the displacement-response curve is of considerable importance in signalling, since it enables the hair cell to evade a limit to response frequency set by its membrane time constant. A

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Consider the response of a cell to a stimulus of period substantially longer than the time constant (Fig. 3). As the hair bundle moves back and forth, a conductance change occurs with a lag of only a few microseconds 3 (Fig. 3A). The transductionchannel current into the cell increases during positive stimulus components and decreases during negative ones, yielding alternate depolarizations and hyperpolarizations. These in turn modulate the release of chemical transmitter by the hair cell and produce a phase-locked pattern of activity in the afferent nerve fiber with which it synapses (Fig. 3B). Imagine instead a stimulus of much higher frequency, up to the 100 kHz to

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Fig. 3. Schematic diagrams depicting the likely chain o f events leading from hair-bundle deflection to firing in an afferent nerve fiber (A) When the hair bundle is deflected by a symmetrical, sinusoidal stimulus ( S), the rectifying property o f the displacement-response relationship results in an asymmetrical change 1°in the transduction conductance (gO. (B) Stimulation at a low frequency (20 Hz) evokes an oscillatory flow o f transduction current (it); the biphasic receptor potential (Vm) produces pulsatile release of synaptic transmitter and phase-locked firing in the 8th-nerve fiber (NS). (C) Because o f rectification in the displacement-response relationship, a moderately high-frequency (2 kHz) tone produces pulses o f inward current during each stimulus cycle whose effects are summed by the membrane's capacitance. The consequent depolarization enhances the release o f synaptic transmitter and produces an increase in the afferent discharge. Note the differing time scales in Band C.

368 which some mammalian cochleas can respond (Fig. 3C). Conductance changes and current flows again rapidly follow the hair bundle's deflection. Now, however, the stimulus period is much briefer than the hair cell's membrane time constant. If the displacement-response relationship were symmetrical, the increase in inward current during the positive stimulus phase would approximately balance the decreased transduction current during negative stimulation; the net change in transduction current would be near zero and no receptor potential would ensue. Because of the asymmetry in the curve, however, the positive component of a sinusoidal stimulus increases the current more than the negative stimulus component reduces it. Each full cycle of stimulation therefore yields a net inward current which is integrated by the membrane's capacitance to produce a steady depolarization, the intracellular summating potential, for as long as the stimulus lasts. The process resembles the temporal summation of postsynaptic potentials upon rapidly repeated stimulation of an input. The steady depolarization then leads to excitation of the afferent nerve ending and protracted firing which, although not locked to the stimulus' phase, nonetheless signals the presence of a high-frequency input. Since the ability of a hair cell to respond to high-frequency stimuli rests upon the rectifying property of the displacement-response relationship, one might anticipate that the cell contrives to operate in the range of hair-bundle positions where the curve is strongly asymmetrical. In hair cells of the bullfTog's sacculus, an adaptation mechanism shifts the displacement-response relationship along the abscissa so as to keep the cell functioning in a rectifying portion of the curve despite the presence of static stimuli that offset the hair bundle 2. It remains to be learned whether this phenomenon is indeed important in generating summating potentials, whether it occurs in other hair cells, and how it Iranspires, D'wectionai sensitivity Much of the selectivity of vestibular hair cells for linear or angular accelerations arises from the geometry of the fluid.filled labyrinth and from the mechanical properties of the otolithic membrane and cupula, the extracelldar struetu~s that couple stimuli to hair-bundle deflections. The hair cells themselves also contribute to stimulus discrimination, however, most importantly through their directional sensitivity. As has already been noted, each cell has an axis of maximal sensitivity to hair-bundle displacement that corresponds to the cell's plane of bilateral symmetry.

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The output of a given hair cell is ambiguous in its meaning to the nervous system. A given receptor potential might be due to a stimulus of a certain amplitude directed along the axis of greatest sensitivity. Because a cell's sensitivity declines as the cosine of the angle by which the stimulus deviates from the axis of symmetry 8,=, the same response would also be evoked by, for example, a stimulus of twice that size at an angle of 60° from this axis. To overcome the ambiguity inherent in the output of each cell, those organs of the vestibular system that must determine the direction as well as the amplitude of a stimulus deploy a battery of hair cells of varying orientations. In the sacculi and utriculi of many species, for example, hair cells are so disposed that their axes of optimal sensitivity range through nearly 360 ° . By examining the outputs of several differently oriented cells in the saccuhis, the central vestibular apparatus can uniquely determine both the amplitude and the direction of any component of linear acceleration in the plane of the organ. The disposition of hair cells in the sacculus and utricuhis is also interesting as an example of the precision of morphogenetic pattern formation. Contiguous hair cells have very similar orientations; there is a gradual shift in axes with very few deviant cells. Moreover, the organ is approximately bisected by a line along which the polarities of the cells abruptly shift by 180 °. Not surprisingly, such elaborate orientation

patterns of hair bundles do not occur m those organs of the acousticolateralis system in which the direction of stimulation is fixed. In semicircular canals and lateralline organs, for instance, fluid is constrained to flow along the axis of the organ; here the hair cells are all arranged with their axes of sensitivity parallel to that of fluid motion 8'~8. In the cochlea, where auditory stimuli are converted into shear between the hair cells and an overlying tectorial membrane, the hair cells are so oriented that their greatest sensitivity lies along the direction of shear24

Frequency tuning The classic work of yon B6k~sy 24 estab. lished a basis for frequency tuning, the capacity of cells in the auditory system to respond best to a relatively narrow range of stimulus frequencies, in the complex mechanical behavior of the mammalian cochlea. Stimuli reaching the cochlea are there resolved into their constituent sinusoidal components, each of which, by effecting a localized vibration of a portion of the basilar membrane 15,19~, stimulates a specific ensemble of hair cells. This particular 'pitch-by-place' mechanism can operate, however, only in the relatively long cochleas of mammals, birds and higher reptiles. Recordings from the 8th nerves of certain lower reptiles and amphibians have revealed comparably sharp frequency selectivity despite the fact that these species lack sharp mechanical tuning of the basilar membrane. How might tuning occur in these animals? In the instance of the red-eared turtle 10 mV there is compelling evidence that a substantial component of the tuning resides in the electrical properties of individual hair cells. Upon being stimulated by the injection of current through an intracellular microelectrode, each of these cells exhibits lightly damped oscillations in membrane potential near its 'best' or 'characteristic" frequency, i.e. that frequency at which the cell is most sensitive to sound stimulation 5. It appears that acoustical inputs of a wide range of frequencies, coarsely filtered by the mechanical properties of the turtle's middle ear, deflect the hair bundle of any given hair cell and excite it. Because of the tuned electrical t , I falter of each cell, however, stimuli of only 100 ms a rather narrow range of frequencies elicit 4. Oscillatory potenaals recorded intracel- large responses. Several questions arise in connection lut~ly from a hair cell o f the bull.frog's sacculus. Constam.au'rem p u ~ of various amp!'m_,~_s with this mechanism of frequency tuning. (lower :races), ~ ' ~ d through the mic.rodectm~ What ionic conductances are responsible u m t for the nxordines, evoke d a n t ~ o~_ "_4~_'.~ns for the electrical resonance phenomenon? in manbrant ~ (~l,*r tmats). S t m ~ u s Cells of the bullfrog's sacculus, which also osc#lattons oecur at 3 1 H z at tbe resting pottntial of "62 mY;as has ~ documented ~ v e l y for show oscillatory membrane potentials (Fig. cells in ate turae ~, depolarizaaon mists the fre- 4), possess at least six conductance mechanisms: the transduction conductance quency o f ~ a n d hy.Dc~:~larigalion r e d o e s it. The traces have been offset vertically for clarity. itselfa, a postsynaptic conductance regu-

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lated by efferent-nerve stimulation, a Ca 2+ flexion with respect to their insertions. The conductance12. ~7, and inwardly rectifying~, mechanical properties of the basilar papilla A-type ~7 and Ca2+-activated ~7 K + conduc- and of individual hair bundles are the tances. More detailed information on the important determinants of frequency seleckinetics and potential- and ion-sensitivity of tivity in this animal. these various mechanisms will be necessary Although it is clear that there are at least to determine which contribute to the electri- three strategies for frequency tuning, based cal resonance. How is the frequency of on basilar-membrane mechanics, electrical oscillation determined? Along the 700/xm resonance, and hair-cell micromechanics, it of the turtle's basilar membrane lie about is less certain whether some ears employ 1 000 hair cells whose characteristic fre- more than one of these mechanisms. There quencies vary monotonically between 30 is no evidence to date of an electrical resoHz and 700 Hz (Ref. 4). What features of nance in mammalian hair cells, but it is the membrane vary to produce this range of conceivable that such a mechanism opertuning: capacitance or the numbers, types, ates sufficiently swiftly to enhance tuning kinetics or sensitivities of channels? Can in those responsive to high frequencies. higher centers of the turtle's auditory sys- Moreover, it may well be that tuning as tem distinguish neural activity due to spon- measured at the basilar membrane reflects taneous oscillations in hair-cell membrane not only the hydromechanical properties of potential, which may reach 10 mV in peak- this structure and of the surrounding fluids, to-peak size4, from that evoked by acousti- but also the mechanical characteristics of cal stimuli? the overlying sensory epithelium, the organ Hearing in the alligator lizard presents an of Corti. The cochlea's mechanical non-. interesting contrast with that in the turtle. linearityx6, its ability actively to emit Although recordings from hair cells of the sound 14, and the metabolic lability of these alligator lizard have also demonstrated features and of tuning suggest a cellular sharp frequency tuning, there is no evi- contribution to tuning. The fact that dence for membrane-potential oscillations stimulation of the efferent nerve supply to of the sort seen in the turtle; it seems that outer hair cells diminishes frequency selecmechanical tuning provides the frequency tivity26 argues that the hair cells themselves selectivity of these hair cells2L The basilar are involved. A most interesting prospect membrane of the lizard moves as a unit for the next several years of research is an regardless of the sound frequency applied. elucidation of the role of hair ceils, not only Where, then, does mechanical tuning in transduction, but in tuning as well. occur? The morphology of the basilar papilla provides a clue~: along the length of Acknowledgements the organ there is continuous variation in The author thanks Mr R. Jacobs for supplying the dimensions of the hair cells and of the the scanning electron micrograph and for preparpapilla as a whole. The lengths of the free- ing the figures, Ms C. Hochenedel for typing the standing hair bundles vary in a particularly manuscript, and Dr T. Holton, Mr R. Jacobs, Mr dramatic manner, from 12/zm at the end of R. S. Lewis and Dr J. M. Nerhonne for critical comments. Research in the author's laboratory is the papilla that responds best to 4-kHz tones supported by National Institutes of Health grant to 31 /zm at the point most sensitive to NS-13154 and by the System Development 900-Hz stimuli. Stroboscopic illumination Foundation. of isolated organs reveals that upon stimulation the basilar papilla sways from side to Readinglist side in a complex, frequency-dependent I Corey, D. P. and Hudspeth, A. J. (1979)Nature manner n. Moreover, as the stimulus fre(London) 281,675-677 quency varies, hair bundles of different 2 Corey, D. P. and Hudspeth, A. J. (1983) J. Neurosci. 3,942-961 lengths experience differing amounts of

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A. J. Hudspeth is at the Departments of Physiology and Otolaryngology, University of California School of Medicine, San Francisco, CA 94143,' USA.