Localization of the Cochlear Amplifier in Living Sensitive Ears

BACKGROUND: To detect soft sounds, the mammalian cochlea increases its sensitivity by amplifying incoming sounds up to one thousand times. Although the cochlear amplifier is thought to be a local cellular process at an area basal to the response peak on the spiral basilar membrane, its location has not been demonstrated experimentally. METHODOLOGY AND PRINCIPAL FINDINGS: Using a sensitive laser interferometer to measure sub-nanometer vibrations at two locations along the basilar membrane in sensitive gerbil cochleae, here we show that the cochlea can boost soft sound-induced vibrations as much as 50 dB/mm at an area proximal to the response peak on the basilar membrane. The observed amplification works maximally at low sound levels and at frequencies immediately below the peak-response frequency of the measured apical location. The amplification decreases more than 65 dB/mm as sound levels increases. CONCLUSIONS AND SIGNIFICANCE: We conclude that the cochlea amplifier resides at a small longitudinal region basal to the response peak in the sensitive cochlea. These data provides critical information for advancing our knowledge on cochlear mechanisms responsible for the remarkable hearing sensitivity, frequency selectivity and dynamic range

Local transfer functions, delay, velocity, and wavelength in a different sensitive cochlea.

<p>Data were collected at longitudinal locations ∼2,650 and ∼2,317 µm with ∼333 µm separation. Allowing a higher peak frequency of 15.0 kHz in panel A, the data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020149#pone-0020149-g003" target="_blank">Figure 3</a> are similar to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020149#pone-0020149-g002" target="_blank">Figure 2</a>, which confirm the existence of magnitude amplification and reduction over the BM region between the two measured locations.</p

Frequency-dependent amplification and reduction and BM sharp tuning.

<p>(A) The BM response at the basal location is presented by vibration amplitude as a function of frequency (dotted curve), and that at the more apical location is shown by the solid curve. The peak frequency of the basal location was higher than that of the more apical site. As the vibration propagated from base to apex, the BM between the two measured locations increased low-frequency responses (upward arrow) and reduced high-frequency responses (downward arrow), resulting in a sharply tuned response at the apical location (solid curve). (B) Phase at the basal location (dotted curve) leaded that at the apical location (solid curve), indicating that waves propagated from base to apex at frequency-dependent speeds. Data were collected at 30 dB SPL from the same sensitive cochlea as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020149#pone-0020149-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020149#pone-0020149-g002" target="_blank">2</a>.</p

Diagrams for measuring basilar membrane vibrations.

<p>(A) Two measured locations on the BM and one on the stapes (red dots). As the wave travels from the base to its BF location (B), the cochlear amplifier increases the BM vibration at a location basal to the BF site (blue bar in panel C). The local transfer function can specifically quantify the functioning of the amplification region between positions A and B. (D) shows a sharp peak at ∼15.3 kHz at low sound levels, which was >1,000 at 20 dB SPL. As the sound level increased, the peak magnitude decreased, and the peak broadened and shifted toward ∼12.0 kHz. (E and H) Growth rates in dB/dB at the more basal (E) and apical (H) locations. (F) The phase lag progressively increased with frequency. The data in panels G–I, measured at the more apical location, are similar to those in panels D–F (allowing for a lower BF). BM_B and BM_A are BM vibration magnitudes at the measured basal and apical locations.</p

Local transfer functions, delay, velocity, and wavelength of basilar membrane vibration.

<p>(A) The response peak at ∼12.0 kHz decreased, broadened, and shifted toward low frequencies with increasing sound level. The magnitude was smallest at ∼17.0 kHz. (B) Phase response was similar to that in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020149#pone-0020149-g001" target="_blank">Figures 1F and I</a> but with a smaller phase lag. The delay from the basal to more apical location increased with frequency (C), while the propagation velocity (D) and wavelength (E) decreased over the same frequency range. Red lines show post-mortem data measured at 40 dB SPL.</p

The relationship between transfer functions and the longitudinal pattern of basilar membrane vibration.

<p>In contrast to the >1,000 gain of the conventional transfer function (thin lines) at the peak frequency, the local transfer function (thick lines) shows a gain of only ∼10 at ∼12.0 kHz and ∼40 dB of reduction at ∼17 kHz in panels A. Response peaks became smaller at 90 dB SPL in panel B. (C) At 20 dB SPL, the highest transmission efficiency was >50 dB/mm at ∼12.0 kHz and the lowest efficiency was <−100 dB/mm at ∼17.0 kHz (thick line). (D) At 90 dB SPL, the response peak at ∼12.0 kHz disappeared and the minimum remained unchanged (thick line). (E) BM response to a 50 dB SPL 11.0-kHz tone increased at the rate of ∼26 dB/mm in the region between 2,450 to 2,750 µm (green arrow), while the 19.0-kHz response decreased at the rate of ∼131 dB/mm over the same distance (red arrow). (F) The increase in low-level response on the basal side of the BF location (solid green arrows) became the decrease at the high sound level (red arrow near 2,300 µm).</p

The group delay and suppression pattern of the cochlear microphonic potential recorded at the round window.

It is commonly assumed that the cochlear microphonic potential (CM) recorded from the round window (RW) is generated at the cochlear base. Based on this assumption, the low-frequency RW CM has been measured for evaluating the integrity of mechanoelectrical transduction of outer hair cells at the cochlear base and for studying sound propagation inside the cochlea. However, the group delay and the origin of the low-frequency RW CM have not been demonstrated experimentally.This study quantified the intra-cochlear group delay of the RW CM by measuring RW CM and vibrations at the stapes and basilar membrane in gerbils. At low sound levels, the RW CM showed a significant group delay and a nonlinear growth at frequencies below 2 kHz. However, at high sound levels or at frequencies above 2 kHz, the RW CM magnitude increased proportionally with sound pressure, and the CM phase in respect to the stapes showed no significant group delay. After the local application of tetrodotoxin the RW CM below 2 kHz became linear and showed a negligible group delay. In contrast to RW CM phase, the BM vibration measured at location ∼2.5 mm from the base showed high sensitivity, sharp tuning, and nonlinearity with a frequency-dependent group delay. At low or intermediate sound levels, low-frequency RW CMs were suppressed by an additional tone near the probe-tone frequency while, at high sound levels, they were partially suppressed only at high frequencies.We conclude that the group delay of the RW CM provides no temporal information on the wave propagation inside the cochlea, and that significant group delay of low-frequency CMs results from the auditory nerve neurophonic potential. Suppression data demonstrate that the generation site of the low-frequency RW CM shifts from apex to base as the probe-tone level increases

Annexin A5 is the Most Abundant Membrane-Associated Protein in Stereocilia but is Dispensable for Hair-Bundle Development and Function

The phospholipid- and Ca2+-binding protein annexin A5 (ANXA5) is the most abundant membrane-associated protein of ~P23 mouse vestibular hair bundles, the inner ear’s sensory organelle. Using quantitative mass spectrometry, we estimated that ANXA5 accounts for ~15,000 copies per stereocilium, or ~2% of the total protein there. Although seven other annexin genes are expressed in mouse utricles, mass spectrometry showed that none were present at levels near ANXA5 in bundles and none were upregulated in stereocilia of Anxa5−/− mice. Annexins have been proposed to mediate Ca2+-dependent repair of membrane lesions, which could be part of the repair mechanism in hair cells after noise damage. Nevertheless, mature Anxa5−/− mice not only have normal hearing and balance function, but following noise exposure, they are identical to wild-type mice in their temporary or permanent changes in hearing sensitivity. We suggest that despite the unusually high levels of ANXA5 in bundles, it does not play a role in the bundle’s key function, mechanotransduction, at least until after two months of age in the cochlea and six months of age in the vestibular system. These results reinforce the lack of correlation between abundance of a protein in a specific compartment or cellular structure and its functional significance

Magnitude of the RW CM as a function of suppressor frequency in a different sensitive cochlea.

<p>The RW CM was evoked by 500-Hz probe tones at 30 to 80 dB SPL, and suppressed by the second tone at different frequencies and levels. For 30-, 40-, and 50-dB probe tones and at intermediate suppressor levels, the RW CM was suppressed dominantly at frequencies below 5 kHz (panels A–C). At 70- and 80-dB SPL probe-tone levels, suppression occurred mainly at high frequencies near 10 kHz. Suppressor-induced CM increase occurred at frequencies near 9 kHz (panels B–E) and below 1 kHz (panel D).</p

Diagram for measuring RW CM, the stapes and BM vibrations.

<p>(A) The CM was measured from the RW niche (red dot). Sound-induced vibrations were measured from the stapes and at a BM location ∼2.5 mm from the base (green dots). The temporal relationships between signals are described by the following delays: the delay from speakers to the stapes (), the forward and backward delays in the cochlea ( and ). (B) The spatial relationship between travelling waves and the electrode locations for recording the RW CM. BM: the basilar membrane; RW: the round window; BF: best frequency.</p