The mechanical sensitivity of hair cells is not constant; responsiveness varies in such a way that a given cell best detects behaviorally relevant stimuli. When it is appropriate that low-frequency inputs be disregarded, hair cells possess a unique mechanism of adaptation that acts as a high-pass filter. In addition, many hair cells in auditory systems display electrical resonance that tunes them to specific frequencies of stimulation. Finally, hair cells employ mechanical amplification that enhances and further tunes their mechanosensitivity.
Hair Cells Adapt to Sustained Stimulation
Despite the precision with which a hair bundle grows, it cannot develop in such a way that the sensitive transduction apparatus is perfectly poised at its position of greatest mechanosensitivity. Some mechanism must compensate for developmental irregularities, as well as for environmental changes, by adjusting the gating springs so that transduction channels are active at the bundle's resting position. An adaptation process that continuously resets the hair bundle's range of mechanical sensitivity does just that. Because of adaptation, a hair cell can maintain a high sensitivity to transient stimuli while rejecting static inputs a million times as large.
Adaptation manifests itself as a progressive decrease in the receptor potential during protracted deflection of the hair bundle (Figure 30–10). The process is not one of desensitization, for the sensitivity of the receptor persists. However, with prolonged stimulation the sensitivity shifts from that of the bundle's resting point to approximately 80% of its deflected position. Adaptation occurs on a time scale three orders of magnitude slower than mechanoelectrical transduction: The time constant of adaptation is approximately 20 ms when endolymph bathes the hair bundle. The rate and extent of adaptation increase with increasing concentration of Ca2+ in the liquid contacting the apical cell surface.
Adaptation of mechanoelectrical transduction in hair cells.
A. The lower trace shows mechanical test stimuli of various sizes applied before prolonged (100 ms) deflection of a hair bundle (1) and at two times during the deflection (2, 3). As shown in the upper trace, the rapid depolarization at the onset of the deflection is followed by a gradual decline toward a plateau. Seven records, each with a different test stimulus, are superimposed.
B. Adaptation alters the relation between hair-bundle displacement and the receptor potential of the hair cell before and during displacement. Each of the three curves is generated by plotting the electrical responses to the seven test stimuli against the respective hair-bundle displacements. As adaptation proceeds the sigmoidal relation shifts to the right along the abscissa without substantial changes in the curve's shape or amplitude. This result implies that during adaptation to a protracted stimulus a hair bundle's range of mechanical sensitivity approaches the position at which the bundle is held.
How does adaptation occur? Because the mechanical force exerted by a hair bundle changes as adaptation proceeds, the process evidently involves an adjustment in the tension borne by the gating springs. It appears likely that the structure anchoring the upper end of each tip link, the insertional plaque, is repositioned during adaptation by an active molecular motor. Hair bundles contain at least five isoforms of myosin, the motor molecule associated with motility along actin filaments (see Chapter 34). Immunohistochemical studies indicate that myosin-1c occurs in clusters at insertional plaques and near the stereociliary tips, and site-directed mutagenesis implicates this isozyme in adaptation. Several dozen such myosin molecules associated with each tip link are thought to maintain tension by ascending cytoskeletal actin filaments and pulling the link's insertion with them (Figure 30–11).
A model of adaptation by hair cells.
Prolonged deflection of the hair bundle in the positive direction elicits an initial depolarization followed by a decline to a plateau and an undershoot at the cessation of the stimulus. Initially the stimulation increases tension in the tip link, thus opening transduction channels. As stimulation continues, however, a tip link's upper attachment is thought to slide down the stereocilium, allowing each channel to close during adaptation. Prolonged deflection of the hair bundle in the negative direction elicits a complementary response. The cell is slightly hyperpolarized at first but shows a rebound depolarization at the end of stimulation; tension is restored to the initially slack tip link as myosin molecules actively pull up the link's upper insertion.
When a stimulus step increases the tension in a gating spring, the associated transduction channel opens, permitting an influx of cations. As Ca2+ ions accumulate in the stereociliary cytoplasm, they bind to the calmodulin light chains that adorn the neck region of myosin-1c. The activation of calmodulin in turn reduces the upward force of the myosin-1c molecule, thereby shortening the gating spring. When the spring reaches its resting tension, closure of the channel reduces the Ca2+ influx to its original level, restoring a balance between the upward force of myosin and the downward tension in the spring.
Hair Cells Are Tuned to Specific Stimulus Frequencies
As a result of the tonotopic arrangement of the mammalian basilar membrane, every cochlear hair cell is most sensitive to stimulation at a specific frequency, termed its characteristic, natural, or best frequency. On average, the characteristic frequencies of adjacent inner hair cells differ by approximately 0.2%; adjacent piano strings, in comparison, are tuned to frequencies some 6% apart.
The sensitivity of a cochlear hair cell extends within a limited range above and below its characteristic frequency. This follows from the fact that the traveling wave evoked even by a pure sinusoidal stimulus spreads somewhat along the basilar membrane. The traveling wave of a stimulus tone with a pitch lower than the characteristic frequency of a particular hair cell passes that cell and peaks somewhat farther up the cochlear spiral. A higher-pitched tone causes a traveling wave that crests below the cell. Nevertheless, in either instance the basilar membrane undergoes some motion at the hair cell's site, so that the cell responds to the stimulus.
The frequency sensitivity of a hair cell may be displayed as a tuning curve. To construct a tuning curve, an experimenter stimulates the ear with pure tones at numerous frequencies below, at, and above the cell's characteristic frequency. The intensity of stimulation is adjusted for each frequency until the response reaches a predefined criterion magnitude. An investigator might, for example, ask what stimulus intensity is necessary at each frequency to produce a receptor potential 1 mV in peak-to-peak magnitude. The tuning curve is then a graph of sound level, presented logarithmically in decibels sound-pressure level, against stimulus frequency.
The tuning curve for an inner hair cell is typically V-shaped (Figure 30–12). The curve's tip represents the cell's characteristic frequency, the frequency that produces the criterion response for the lowest- intensity stimulus. Sounds of greater or lesser frequencies require higher intensities to excite the cell to the criterion response. As a consequence of the traveling wave's shape, the slope of a tuning curve is far steeper on its high-frequency flank than on its low-frequency flank.
Tuning curves for cochlear hair cells.
To construct a curve the experimenter presents sound at several frequencies. At each frequency the stimulus intensity is adjusted until the cell produces a criterion response, here 1 mV. The curve thus reflects the threshold of the cell for stimulation at a range of frequencies. Each cell is most sensitive to a specific frequency, its characteristic frequency. The threshold rises briskly—the sensitivity falls abruptly—as the stimulus frequency is raised or lowered. (Reproduced, with permission, from Pickles 1988.)
Hair cells must contend with acoustic stimuli that have a very low energy content. If the stimulus consists of a periodic signal, such as the sinusoidal pressure of a pure tone, a detection system can increase the signal-to-noise ratio by enhancing selectively the response to a relevant frequency. At least two cellular mechanisms are known to accomplish this task, thereby supplementing the tuning accomplished by the basilar membrane.
First, the mechanical properties of a hair bundle help tune it to a particular frequency, in the same way a tuning fork's resonant frequency depends on the mechanical properties of its tines. These properties include the bundle's flexibility and mass. The flexible elements that restore a bundle to its resting upright position are the gating springs and the actin-filled rootlets at the base of the stereocilia. Because the bundle moves through a viscous medium, the mass relevant to the bundle's tuning includes that of a volume of water dragged along by the moving bundle. Viscosity also heavily dampens the motion.
In the auditory organs of many animals the lengths of the hair bundles vary systematically along the tonotopic axis. Hair cells that respond to low-frequency acoustic stimuli have the longest bundles, whereas those that respond to the highest-frequency signals bear the shortest bundles. In the human cochlea, for example, an inner hair cell with a characteristic frequency of 20 kHz bears a 4 μm hair bundle. At the opposite extreme, a cell sensitive to a 20 Hz stimulus has a bundle more than 7 μm high.
Computer modeling shows that the tuning-fork mechanism helps to tune cochlear hair cells with freestanding hair bundles, those that are not attached to a tectorial membrane. In humans these cells are the inner hair cells, the receptors that provide most of the information conveyed by the cochlear nerve. The length of the stereocilia may also affect the tuning of cells whose hair bundles are inserted into a tectorial membrane, for in these hair cells too there is an inverse relation between bundle length and characteristic frequency.
The second mechanism that tunes individual hair cells to specific frequencies is electrical in nature. In many fishes, amphibians, and reptiles including birds the membrane potential of each hair cell resonates at a particular frequency in response to an injected current pulse (Figure 30–13A). When a cell is stimulated with sounds of various frequencies but constant amplitude, it responds over a broad range of frequencies. However, when current is injected the cell responds most strongly to stimulation at the particular frequency at which the cell's membrane potential resonates. Whether electrical resonance contributes to frequency tuning in the ears of mammals, including humans, remains uncertain.
Electrical tuning of a hair cell.
A. 1. When the hair bundle is deflected, this hair cell's membrane potential oscillates at a frequency of approximately 180 Hz. 2. Passing electrical current into the same hair cell through a microelectrode evokes oscillation of the membrane potential at a similar frequency, an indication that the cell is tuned to a specific stimulus frequency by an electrical resonator. (Reproduced, with permission, from Crawford and Fettiplace 1981.)
B. A model of electrical resonance in a hair cell. Positive deflection of the hair bundle opens mechanoelectrical transduction channels in the stereocilia, thus depolarizing the cell. The depolarization opens voltage-sensitive Ca2+ channels, and the resulting Ca2+ influx augments the depolarization. As Ca2+ accumulates in the cytoplasm, however, it activates Ca2+-sensitive K+ channels that, along with voltage-sensitive K+ channels, allow K+ efflux that repolarizes the cell. To maintain an appropriate cytoplasmic Ca2+ concentration the Ca2+ must be sequestered and eventually pumped from the cell.
The basis of electrical resonance has been determined by voltage-clamp recordings from isolated hair cells. The depolarizing phase of an oscillation is driven by current carried into the cell through voltage-gated Ca2+ channels, whereas the repolarizing component results primarily from outward current through Ca2+-sensitive K+ channels (Figure 30–13B). Several factors establish the frequency and sharpness of the resonance, including the membrane capacitance, the numbers of Ca2+ and K+ channels, and the time course of Ca2+ removal. In addition, variation in the K+ channels along the cochlea is a factor in the differences in frequency selectivity of the hair cells. Alternative splicing of the mRNA encoding cochlear K+ channels generates several channel isoforms that differ in their kinetics and their sensitivities to Ca2+ and voltage. Moreover, expression of the channel's auxiliary β subunit, which also regulates gating kinetics, displays a gradient along the tonotopic axis. How hair cells become tuned to their characteristic frequencies during development remains to be determined.
Sound Energy Is Mechanically Amplified in the Cochlea
The inner ear faces an important obstacle to efficient operation: A large portion of the energy in an acoustic stimulus goes into overcoming the damping effects of cochlear liquids on basilar-membrane motion rather than into excitation of hair cells. The sensitivity of the cochlea is too great, and auditory frequency selectivity too sharp, to result solely from the inner ear's passive mechanical properties. The cochlea must therefore possess some means of actively amplifying sound energy.
One indication that amplification occurs in the cochlea comes from measurements of the basilar membrane's movements with sensitive laser interferometers. In a preparation stimulated with low-intensity sound the motion of the membrane at any point is highly sensitive to frequency. As the sound intensity is increased, however, the membrane's sensitivity declines precipitously and its tuning becomes less sharp: The sensitivity of basilar-membrane motion to stimulation at 80 dB is less than 1% that for 10 dB excitation. The sensitivity predicted in modeling studies of a passive cochlea corresponds to that observed with high-intensity stimuli. This result implies that the motion of the basilar membrane is augmented more than 100-fold during low-intensity stimulation but that amplification diminishes progressively as the stimulus grows in strength.
In addition to this circumstantial evidence, experimental observations support the idea that the cochlea contains a mechanical amplifier. When a normal human ear is stimulated with a click, that ear emits one to several measurable pulses of sound. Each pulse includes sound in a restricted frequency band. High-frequency sounds are emitted with the shortest latency, approximately 5 ms, whereas low-frequency emissions occur after a delay as great as 20 ms (Figure 30–14A). These so-called evoked otoacoustic emissions are not simply echoes; they represent the emission of mechanical energy by the cochlea, triggered by acoustic stimulation.
The cochlea actively emits sounds.
A. The records display evoked otoacoustic emissions from the ears of five human subjects. A brief click was played into each ear through a miniature speaker. A few milliseconds later a tiny microphone in the external auditory meatus detected one or more bursts of sound emission from the ear. (Reproduced, with permission, from Wilson 1980.)
B. Under suitably quiet recording conditions, spontaneous otoacoustic emissions occur in most normal human ears. This spectrum displays the acoustic power of six prominent emissions and several smaller ones. (Reproduced, with permission, from Murphy et al. 1995.)
A still more compelling manifestation of the cochlea's active amplification is spontaneous otoacoustic emission. When a suitably sensitive microphone is used to measure sound pressure in the ear canals of subjects in a quiet environment, at least 70% of normal human ears continuously emit one or more pure tones (Figure 30–14B). Although these sounds are generally too faint to be directly audible by others, physicians have reported hearing sounds emanating from the ears of newborns! The ears of adults, too, occasionally emit audible sounds. The active process in the cochlea ordinarily serves to counter the viscous damping effects of cochlear fluids on the basilar membrane. However, if this cochlear amplifier is overly active the ear emits sound, just as a public-address system howls when its gain is excessive.
What is the source of evoked and spontaneous otoacoustic emissions, and presumably of cochlear amplification as well? Several lines of evidence implicate outer hair cells as the elements that enhance cochlear sensitivity and frequency selectivity and hence as the energy sources for amplification. The afferent nerve fibers that extensively innervate the inner hair cells make only minimal contacts with the outer hair cells. Instead, the outer hair cells receive an extensive efferent innervation that, when activated, decreases cochlear sensitivity and frequency discrimination. Pharmacological ablation of outer hair cells with selectively ototoxic drugs degrades the ear's responsiveness still more profoundly.
When stimulated electrically, an isolated outer hair cell displays the unique phenomenon of electromotility: The cell body shortens when depolarized and elongates when hyperpolarized (Figure 30–15). This response can occur at frequencies exceeding 80 kHz, an attractive feature for a process postulated to assist high-frequency hearing.
Voltage-induced motion of an outer hair cell.
Depolarization of an isolated outer hair cell through the electrode at its base causes the cell body to shorten (left); hyperpolarization causes it to lengthen (right). The oscillatory motions of outer hair cells may provide the mechanical energy that amplifies basilar-membrane motion and thus enhances the sensitivity of human hearing. (Reproduced, with permission, from Holley and Ashmore 1988.)
The energy for these movements is drawn from the experimentally imposed electrical field rather than from hydrolysis of an energy-rich substrate such as adenosine triphosphate (ATP). Movement occurs when changes in the electric field across the membrane reorient molecules of the protein prestin. The concerted movement of several million of these molecules changes the membrane's area and thus the cell's length. When an outer hair cell transduces mechanical stimulation of its hair bundle into receptor potentials, cochlear amplification might then occur as voltage-induced movement of the cell body augments basilar-membrane motion. Consistent with this hypothesis, mutation of certain amino-acid residues required for the voltage sensitivity of prestin abolishes the active process in mice.
Because sharp tuning, high sensitivity, and otoacoustic emissions are also observed in animals that lack outer hair cells, electromotility cannot be the only form of mechanical amplification by hair cells. In addition to detecting stimuli, hair bundles are also mechanically active and contribute to amplification. Hair bundles can make spontaneous back-and-forth movements that might underlie spontaneous otoacoustic emissions. Under experimental conditions bundles can exert force against stimulus probes, performing mechanical work and thereby amplifying the input. In vitro experiments indicate that active hair-bundle motility contributes to the cochlear active process even in the mammalian ear.
Several features of auditory responsiveness suggest that cochlear hair cells operate on the verge of an instability termed the Hopf bifurcation. This phenomenon explains hair cells' amplification and frequency selectivity, their nonlinear sensitivity to stimulus intensity, and their capacity to become unstable and spontaneously emit sound. The fact that active hair-bundle motility demonstrates a Hopf bifurcation in vitro provides further evidence that this mechanism contributes to cochlear amplification.
Although active hair-bundle movements have been demonstrated at sound frequencies as high as a few kilohertz, it remains uncertain whether bundles can generate forces at the very high frequencies at which sharp frequency selectivity and otoacoustic emissions are observed in the mammalian cochlea. Active hair-bundle motility and electromotility may function synergistically, with the former serving metaphorically as a tuner and preamplifier and the latter as a power amplifier. Alternatively, hair-bundle motility may operate at relatively low frequencies but be superseded by electromotility at the highest frequencies.