Auditory Periphery: Cochlear Processing Fabio Mammano Laboratory of Biophysics, International School for Advanced Studies, Trieste, Italy ‘Mammano F. Auditory periphery: cochlear processing. Scand Audiol 1997:26 (Suppl 46):5-8, Key words: Basilar membrane, cochlea, hair cell, hearing, sound. Address for offprints: Fabio Mammano, SISSA, Via Beirut, 2/4, 34014 Trieste, Italy Introduction For over 50 years, propagation of travelling waves on the basilar membrane of the cochlea has been known to underlie generation of the auditory sensation (von Békésy, 1960). Physiology textbooks so far give only a graphical representation of this phenomenon, stressing action potential generation on the afferent acoustic nerve fibres contacting inner hair cells. These produce a receptor potential upon displacement of their stereocilia bundle: a process called forward transduction. Stereo- cilia deflection is caused by the shearing motion of the tectorial membrane relative to the organ of Corti, which rides on the basilar membrane as the wave pushes it up and down. In this type of representation the organ of Corti has no internal degrees of freedom. Besides, the fact that the more numerous outer hair cells are prevalently contacted by efferent innervation (Spoendlin, 1978) is completely overlooked. At the beginning of the 1980s crucial experiments performed on the guinea pig basilar membrane showed that travelling wave amplitudes are several orders of magnitude larger in vivo than post-mortem, or even post-trauma (Sellick et al., 1982, 1983; Johnstone et al., 1986). The wave amplitude profiles are also rather different in a properly functioning cochlea, being much mote peaked and terminating abruptly after reaching their maximum, When the wave amplitude is plotted against stimulus frequency at a fixed site on the basilar membrane, the slope on the falling high-frequency skirt is hundreds of dB/octave for near-threshold acoustic inputs. Davis (1983) called “cochlear amplifier’’ the physiologically vulnerable process that underlies travel- ling wave amplification. A cochlea with amplification is said to be active, in contrast to the passive one typical of von Békésy’s description. Here I discuss some key results concerning the mechanisms of travelling wave amplification in an active cochlea. The Cochlear Amplifier The quest for the cochlear amplifier fired a spate of research world-wide. Outer hair cells isolated in a dish were found to respond to extracellular sinusoidal low- frequency electric fields with cycles of contraction! elongation (Brownell et al., 1985). Motile responses could be elicited at acoustic frequencies by changing the transmembrane voltage under whole-cell patch-clamp conditions (Ashmore, 1987). The conversion of elec- trical stimuli into motile responses was called reverse transduction, and the equation cochlear-amplifier cell- motility readily established. But how exactly can cell motility affect the propagation of travelling waves in the cochlea? The basic requirement for amplification is that the forces generated by the outer hair cells be large enough to move the basilar membrane, We tested this hypothesis in ‘an isolated cochlea preparation, perfused with oxygenated artificial perilymph, using a displacement- sensitive interferometer. The outer hair cells were stimulated with current passed across the intact cochlear partition. We found that the organ of Corti distorted under the action of electrically-driven cell length changes and produced place-specific vibration of the basilar membrane with magnitude comparable to that observed near auditory threshold. These experiments supplied the first direct evidence that cochlear amplifica- tion arises from the properties of the outer hair cell population. We also demonstrated that each transversal section of the partition possesses at least one internal degree of freedom, as the top of the organ of Corti, the Scand Audiol 26, Sp 466 F Mammano a8meyD (9) Burum puewutos smoys aus doy, 1asuo pueww0s ON JNU Q] Jo 90uUasa1d 24 qued gw9]yooo 1Oe UL aep UT laud ¥a[4909 parejost 2up [Aw] penuejod eueiquew 0g 0 0z- Ov 09- jueweoe|dsp eBeyo enneley 9 sTBY J2INO UE 0 eousseyig 01 duteja-yoned v (pis por vor 1ayod puewaro (doy *y) “are jo way “1 Sky areiAoues-eN Ww OL g joquog, or AW EL SUA Vv Scand Audiol 26, Sup 46reticular lamina, and the basilar membrane moved in antiphase when the cells were stimulated (Mammano and Ashmore, 1993). Inside the Motor ‘The normal pattern of motion of the basilar membrane in, response to current passed across the cochlear partition was a damped oscillation of up to Snm at the characteristic frequency appropriate for the recording site (Mammano & Ashmore, 1995). When 10mM sodium-salicylate was added to the perfusion, the electrically-evoked basilar-membrane motion was dras- tically but reversibly reduced within minutes (Mammano & Ashmore, 1993; Ashmore et al., 1995). Noticeably, salicylates (aspirin) are known to induce hearing threshold elevation (Douek et al., 1983), reduction of otoacoustic emissions (McFadden & Plattsmier, 1984) and tinnitus. When outer hair cells in the same preparation were patch-clamped under direct visual inspection, measur- able changes in length could be elicited by rapidly changing the cell potential. For typical depolarizing commands of between 50 and 100 mV from rest, the cell shortened by up to 1.5 ym. An appreciable displacement of neighbouring cells was also apparent as distortions of the cochlear partition induced by stimulating a single cell were mechanically propagated over a range of 10-20 um on each side of the cell. The largest potential-induced displacements were found towards the cell base, whereas virtually no motion was observed near the pipette tip, ‘The sensitivity to applied potential steps was 12 nm/mV (Mammano et al., 1995). Voltage-dependent fast transient currents, lasting 1 ms or less, were present at onset and offset of the command steps (Fig. 1A, top). Superfusion with 10 mM salicylate reversibly abolished the transients (Fig. 1A, bottom) and suppressed the motile responses. Subtracting the currents obtained in the presence of salicylate from controls eliminated the contribution to the transients from leakage and ionic currents (Fig, 1B), Integration of the difference current yielded a charge transfer with sigmoidal dependence upon membrane potential (Fig. 1C) that mirrored closely the voltage dependence of the cell length changes (Ashmore, 1987). The dose/charge curve measured during slow bath application of salicylate in isolated cells had a Hill coefficient of 3.4, a half-maximal dose of 3.9 mM and did not show appreciable voltage dependence (Ashmore et al., 1995). The cell membrane is known to be densely packed Auditory periphery 7 with 12 nm protein particles which are believed to be organized in tetramers (Kalinec et al., 1992) and have been indicated as the molecular substrate of the outer hair cell motor (Dallos et al., 1991). Cell length changes would thus be associated with a reorganization of the tetramers in the plane of the membrane powered by the transmembrane electric field and revealed as fast charge transients. A possible explanation for the effects of salicylate and the voltage independence of its block is that salicylate molecules, which possess both lipophilic and hydrophilic properties, wedge through the mem- brane and impede the rearrangement of the motor proteins. In normal conditions the proteins are instead free to drive the cell actin-spectrin cytoskeletal network (cortical lattice), to which they are connected by 25 nm long pillars (Holley & Ashmore, 1988), converting surface into axial forces (Fig. 2) Modelling From a physico-mathematical point of view the organ of Corti can be seen as a set of adjacent segments about 10m long formed by transversal portions of the cochlear partition, each having the structure of a local Resting ae Depolarized Salicylate Fig. 2, Motor inner workings. (Left) Schematic drawing of an outer hair cell. Horizontal arrow indicates displacement of the stereocilia bundle in the excitatory (depolarizing) direction. The cilia are connected by tip links. Inthe cochlea, mechanical input to the stereocilia comes from the overlying tectorial membrane. Stereocilia deflection stretches the tip links inducing the opening of mechanosensitive cation channels attached to the links. Channel gating modulates the resistance of the cell apical membrane, resulting in the ‘generation of a receptor potential (forward transduction). The cell responds by changing length (vertical arrows, reverse transduction). (Right) Motor protein particles inthe cell membrane are connected to the cytoskeleton by short pillars (top). Depolarization induces a conformational change in the particle distribution, diminishing the cel surface. Interaction with the cytoskeleton converts surface forces to axial forces applied along the cell length (botom left). The transconformation is blocked by salicylate molecules across the membrane (bottom right) Scand Audiol 26, Sup 45