Hair Cell Transduction, Tuning, and Synaptic Transmission in the Mammalian Cochlea

Abstract

Sound pressure fluctuations striking the ear are conveyed to the cochlea, where they vibrate the basilar membrane on which sit hair cells, the mechanoreceptors of the inner ear. Recordings of hair cell electrical responses have shown that they transduce sound via submicrometer deflections of their hair bundles, which are arrays of interconnected stereocilia containing the mechanoelectrical transducer (MET) channels. MET channels are activated by tension in extracellular tip links bridging adjacent stereocilia, and they can respond within microseconds to nanometer displacements of the bundle, facilitated by multiple processes of Ca2+-dependent adaptation. Studies of mouse mutants have produced much detail about the molecular organization of the stereocilia, the tip links and their attachment sites, and the MET channels localized to the lower end of each tip link. The mammalian cochlea contains two categories of hair cells. Inner hair cells relay acoustic information via multiple ribbon synapses that transmit rapidly without rundown. Outer hair cells are important for amplifying sound-evoked vibrations. The amplification mechanism primarily involves contractions of the outer hair cells, which are driven by changes in membrane potential and mediated by prestin, a motor protein in the outer hair cell lateral membrane. Different sound frequencies are separated along the cochlea, with each hair cell being tuned to a narrow frequency range; amplification sharpens the frequency resolution and augments sensitivity 100-fold around the cell's characteristic frequency. Genetic mutations and environmental factors such as acoustic overstimulation cause hearing loss through irreversible damage to the hair cells or degeneration of inner hair cell synapses. © 2017 American Physiological Society. Compr Physiol 7:1197-1227, 2017.

Figure 1

Schematic of the sound transmission pathway from the eardrum to the cochlea. Sound stimuli impinge on the tympanum (t), or eardrum, at the end of the ear canal and the vibrations (denoted by red arrows) are transmitted through the three bones of the middleear: malleus (m), incus (i) and stapes (s). The footplate of the stapes behaves like a piston in the oval window and initiates pressure waves in the cochlear fluids so setting in vibration the basilar membrane. The pressure is relieved at the round window (rw). The cochlea, here depicted as straight, is in situ coiled like a snail’s shell and embedded in the petrous temporal bone. It is sub-divided into three compartments containing perilymph or endolymph fluid, the two outer compartment being connected by the helicotrema. The total length of the cochlea is 35 mm (humans), 26 mm (cat), 18 mm (guinea pig), and 6 mm (mice).

Figure 2

Cross section though the cochlear duct showing the cellular structure. The scala media is delimited by Reissner’s membrane, the spiral ligament and the basilar membrane which is surmounted by the organ of Corti. The width of the basilar membrane ranges from approximately 100 to 500 µm in humans. The scala media is filled with a K⁺-based endolymph, here colored pink. The organ of Corti contains the sensory hair cells embedded in assorted supporting cells of distinct shape. The hair-cell stereociliary bundles are covered in an acellular tectorial sheet and the cells are innervated by the cochlear branch of the VIIIth cranial nerve. Inner hair cells are contacted by afferents (orange) whereas outer hair cells are innervated mainly by efferent fibers (yellow). The stria vascularis is an epithelial strip on the lateral wall that is specialized for secreting endolymph.

Figure 3

Schematic of the stria vascularis. The stria comprises two cellular layers separated by an intrastrial space. Marginal cells face the endolymph and intermediate/basal cells, interconnected by gap junctions (blue pairs of lines), are exposed to fibrocytes of the strial ligament and perilymph; adjacent cells in each layer are linked by tight junctions (purple). (Note that the orientation is reversed with regard to that shown in Figure 2.) Flow of K⁺ ions is facilitated by the inwardly-rectifying KCNJ10 K⁺ channel on intermediate cells and the KCNQ1/KCNE1 K⁺ channel on the endolymphatic aspect of the marginal cells. Ionic balance is maintained by Na/K ATPase, Na-2Cl-K and Cl transporters. The voltages given (+90, +100, +10 mV) refer to the static potentials of the extracellular spaces with respect to the scala tympani. The endolymphatic potential of +90 mV is attributable to a Nernst K⁺ equilibrium potential of ~100 mV across the highly K⁺ selective apical membrane of intermediate cells. The intrastrial space has low K⁺ due to uptake of the ion by the Na-2Cl-K cotransporter and the Na/K ATPase and K⁺ is then secreted into endolymph across the K⁺-selective membrane of marginal cell.

Figure 4

Stereociliary bundles and the transduction apparatus. Scanning electron micrographs of stereocilary bundles of A, an outer hair cell and B, an inner hair cell, showing the staircase in heights of the rows. C. Transmission electron micrograph of an outer hair cell showing a tip link connecting two stereocilia; the insertion sites of the tip link (TL) are heavily electron dense suggesting dense protein densities. D. Schematic of the molecular structure of the tip link apparatus deduced from various mutations. USH-1 and USH-2 denote different Usher type 1 and type 2 mutations. The association between the N-termini of protocadherin-15 and cadherin-23 is Ca²⁺ dependent. Two MET channels (red) are situated at the lower end of the tip link and are present as complexes with TMIE, LHFPL5, TMC1 and possibly other proteins. Modified from (80)

Figure 5

Mechano-electrical transducer (MET) currents in outer hair cells. A. Schematic of the stimulating and recording techniques. OHCs are patch clamped and the stereociliary bundle is deflected either by a glass probe attached to a piezoelectric device or by a fluid jet. Displacement of the bundle are calibrated by projection of image onto a photodiode array (55, 243). B. MET currents for family of step displacements, X, of a hair bundle, displaying rapid rise to peak and then adaptive decline to a steady level. C. Plot of peak MET current against bundle displacement with an operating range of ~0.25 µm. D. Expanded scale of MET current onset showing that it develops as quickly as the displacement step (shown above) but then adapts with a time constant, τ_A, of 100 µs. E. MET currents in OHCs from the apex and base of the cochlea for sinusoidal modulation of hair bundle position (top). Bundle motion was calibrated by projecting its image on to a pair of photodiodes, the noisy grey trace denoting the photocurrent. F. MET current increases from apex to base of cochlea; current amplitude was 50 percent larger in the reduced Ca²⁺ of the endolymph solution bathing bundle. All currents measured at a holding potential of −84 mV. Modified from (80) (146).

Figure 6

Single MET channels in mouse hair cells. A. Apical outer hair cell: four representative single channel records for 150 nm hair bundle displacement steps; middle, ensemble average of 10 responses; bottom amplitude histograms giving mean single-channel current of 6.2 pA. B. Basal outer hair cell: four representative single channel records for 150 nm hair bundle stimuli; middle, ensemble average of 10 responses; bottom, amplitude histograms giving mean single-channel current of 12 pA. C. Single-channel current and conductance (mean ± 1 SD) as a function of position in the cochlea, expressed as relative distance from the apical end. Total length of cochlea is 6 mm. All measurements made at room temperature and −84 mV holding potential. Modified from (23)

Figure 7

Adaptation assayed with two-pulse experiment. A. MET currents for two series of brief bundle displacements, the first are control steps and the second are test steps, which are preceded by a long adapting step. Note the current decay during the adapting step. B. Current-displacement relationships for first (control) pulse and for second (test) pulse after adapting step. The current I is scaled to its maximum value, I_max. Note the positive shift, ΔX_0.5, in the current-displacement relationship. C. Schematic of experiment where the amplitude of the adapting step was varied. D. Plot of shift in current-displacement relation, ΔX_0.5, as a function of the size of the adapting step. The slope is typically 0.5 – 0.6. All currents measured in outer hair cells at a holding potential of −84 mV. Results from reference (18)

Figure 8

Tonotopic variations in membrane properties of rodent outer hair cells. A. Principal membrane currents determining potential of outer hair cell. MET current, I_MT, carried mainly by K⁺ ions, flows in through MET channels down a potential gradient determined by the positive endolymphatic potential (EP, 90 mV) and the resting potential (V_R, ~ −50 mV); the K⁺ current exits mainly via GK,n channels in lateral wall, down a K⁺ concentration gradient into the perilymph. B. MET conductance, GMT, increases with the characteristic frequency at the location of the hair cell. C. Voltage-dependent K+ conductance, GK,n, increases with hair-cell characteristic frequency. D. Membrane capacitance decreases with hair-cell characteristic frequency, signifying a progressive decrease in the size, mainly the length, of the outer hair cell. Combining results in B, C and D, implies a significant reduction in the membrane time constant determined by C/(G_MET + G_Kn). Results are combined measurements from gerbils (filled circles) and rats (filled squares) and were taken from (129). E. OHC length (and hence membrane area and electrical capacitance) decreases with increase in characteristic frequency in different mammals: a, chinchilla, human; b, guinea pig; c, chinchilla, gerbil; d, guinea pig, chinchilla; e, gerbil, rat; f, chinchilla, mouse, rat; g, guinea pig, rat, human; g, rat, bat; i, mouse; j, bat. Data from [(60); rat, bat, guinea pig, gerbil], [(25); chinchilla], [(239) (235); human], and from author’s laboratory (rat, mouse, gerbil).

Figure 9

Filtering of receptor potentials by inner hair cell. A. Changes in IHC membrane potential elicited by current pulses of magnitudes given next to each trace in isolated guinea pig inner hair cell. Note the voltage inactivation for larger responses. B. Schematic of organ of Corti showing the IHC and innervation by multiple afferents. The medial and lateral sides of the IHC are often referred to as ‘modiolar’ and ‘pillar’, the orientation of which is shown beneath the schematic. C. Receptor potentials in an inner hair cell of an anesthetized guinea pig for tones of different frequencies, given in Hz alongside the traces. At low frequencies, the response is purely sinusoidal, reflecting the sound stimulus. At frequencies above 1000 Hz, the periodic (AC) component is filtered by the membrane time constant leaving a sustained depolarizing (DC) component. D. Synchronization index, indicating phase-locking in auditory nerve discharge, as a function of the frequency of the sound stimulus in auditory nerve fibers of cats (crosses) and guinea pigs (filled and open squares). An index of 1.0 denotes perfect synchronization of the spikes to a specific phase on every cycle of the tone, whereas an index of 0 denotes no relationship between the spike firing and the sound cycle. Records in (A) modified from (153) and (C) and (D) from (222). See also Figure 14 for examples of phase locking.

Figure 10

Tonotopic organization of the turtle auditory papilla. Left, medial view with the hair-cell papilla on the right-hand side of the basilar membrane; scale bar = 100 µm. Right, examples of electrical resonance in hair cells at different positions along the epithelium. Resonant frequency, given beside traces, increases from apex to base. Each record is the voltage response to a small depolarizing current step, the timing of which is shown at top; cells had resting potentials in the range −44 to −51 mV. From (246)

Figure 11

Mechanical and electrical tuning curves in the mammalian cochlea. A. Solid curves are frequency-threshold tuning curves for two auditory nerve fibers in the chinchilla cochlea, with characteristic frequencies of 0.4 and 9.5 kHz. Superimposed on each nerve-fiber tuning curve at similar locations are the basilar membrane vibrations: iso-displacement response (dotted curves, 1-nm left and 2.7 nm right) and isovelocity response (dashed curves, 2.5 µm/s left, and 164 µm/s right). The results indicate almost all of the frequency tuning is present in the basilar membrane vibrations, with isovelocity responses giving better fits to the nerve fiber frequency-threshold curve; from (254). B. Schematic of auditory nerve fiber tuning curves for the cat cochlea based on results in references (164) (125). Similar sets of tuning curves are also available for other mammals including the Mongolian gerbil (215) and the mouse (283).

Figure 12

Outer hair cell contractility mediated by prestin. A. Schematic of outer hair cell with prestin molecules in lateral wall. Force applied to hair bundle open MET channels, causing depolarization and cell contraction due to change in conformation of prestin. B. Transmission electron micrograph of rat outer hair cell immunolabeled for prestin shows gold particles in the lateral wall; abbreviations: st, stereociliary bundle; cp cuticular plate, cy, cytoplasm, jc junctional complex. C. Contractions of outer hair cell evoked by voltage steps from −120 mV to +50 mV; length change measured with dual photodiode; D. Plots of length change in outer hair cell recorded with chloride-based and sulfate-based intracellular solutions. With chloride, the prestin was half-activated at −50 mV, but sulfate shifted the activation relationship ~150 mV positive. B, from (176); C, D, from (140).

Figure 13

Deformation of organ of Corti during stimulation. A. Excitatory (rarefaction) sound stimulus causes upward deflection of basilar membrane and organ of Corti. On conventional view, the entire organ moves upward without changing shape and causes abneural displacement of hair bundles; brown background denotes resting position and black outline new stimulated position. B. Electrical stimulation elicits contraction of outer hair cells and compression of the organ of Corti, with the reticular lamina being pulled down and basilar membrane pulled up. During normal stimulation it is envisage that both processes in A and B will occur sequentially but the exact timing is still uncertain.

Figure 14

Synaptic potentials and action potentials in an auditory afferent. A. Microelectrode recordings from an auditory nerve terminal in the turtle cochlea showing the spontaneous synaptic potentials and action potentials in the absence of a sound stimulus (top) and the response evoked by a tone at 265 Hz, 54 dB SPL (bottom). B. Peristimulus histograms showing phase locking of action potentials to a 265 Hz tone (top) and a 520 Hz tone (bottom) from cell in (A); modified from reference (54).

Figure 15

The synapse between the inner hair cell and cochlear afferent fiber. A. Inner hair cell makes synaptic contacts with multiple (–20) afferent fibers on its basolateral aspect, each synapse having one presynaptic ribbon (blue) and release site onto one afferent. Fibers synapsing on the pillar side are thought to have low thresholds and high resting spontaneous firing; fibers synapsing on the modiolar side have high threshold and low spontaneous discharge. The ribbons are smaller and the post-synaptic glutamate receptor densities (blue strip) are larger for the low threshold fibers. B, Enlargement of the (blue) ribbon surrounded by halo of (yellow) synaptic vesicles. The ribbon is composed of ribeye and piccolo proteins and anchored to the membrane of the release site by bassoon. Vesicles are exocytosed by Ca²⁺ influx through Cav1.3 Ca²⁺ channels on presynaptic membrane and glutamate neurotransmitter binds to GluA2/3 receptors on the post-synaptic membrane. C. High power view of synaptic vesicle, glutamate transporter Vglut3, and Ca²⁺ sensor otoferlin with six C2 domains. D. Conventional view of the life cycle of the synaptic vesicle, from docking at the release site, interaction of vesicular and target SNARE proteins and priming for release, fusion and reuptake. Several of these processes are thought to be Ca²⁺-sensitive and possibly mediated by otoferlin.