The Spatial Origins of Cochlear Amplification Assessed by Stimulus-Frequency Otoacoustic Emissions

Journal Pre-proof The Spatial Origins of Cochlear Amplification assessed by Stimulus Frequency Otoacoustic Emissions Shawn S. Goodman, Choongheon Lee, John J. Guinan, Jr., Jeffery T. Lichtenhan PII:

S0006-3495(19)34431-5

DOI:

https://doi.org/10.1016/j.bpj.2019.12.031

Reference:

BPJ 10243

To appear in:

Biophysical Journal

Received Date: 2 August 2019 Accepted Date: 27 December 2019

Please cite this article as: Goodman SS, Lee C, Guinan Jr JJ, Lichtenhan JT, The Spatial Origins of Cochlear Amplification assessed by Stimulus Frequency Otoacoustic Emissions, Biophysical Journal (2020), doi: https://doi.org/10.1016/j.bpj.2019.12.031. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Biophysical Society.

The Spatial Origins of Cochlear Amplification assessed by Stimulus Frequency Otoacoustic Emissions

Shawn S. Goodman1 Choongheon Lee2, John J. Guinan Jr3,4, Jeffery T. Lichtenhan2,5

1

University of Iowa Communication Sciences and Disorders Iowa City, Iowa, USA.

2

Washington University School of Medicine in St. Louis Department of Otolaryngology St. Louis, Missouri, USA.

3

Harvard Medical School Department of Otolaryngology Boston, Massachusetts, USA.

4

Eaton-Peabody Laboratories Massachusetts Eye and Ear Boston, Massachusetts, USA.

5

Corresponding author Jeffery T. Lichtenhan Department of Otolaryngology, Box 8115 Washington University School of Medicine in St. Louis 660 South Euclid Avenue Saint Louis, MO 63110 USA [email protected] Tel: 1-314-362-7565 FAX: 1-314-362-0315

Running title: Spatial Origin of Cochlear Amplification

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Abstract Cochlear amplification of basilar membrane (BM) traveling waves is thought to occur between a tone’s characteristic frequency (CF) place and within 1 octave basal of CF. Evidence for this view comes only from the cochlear base. Stimulus-frequency otoacoustic emissions (SFOAEs) provide a noninvasive alternative to direct measurements of cochlear motion that can be measured across a wide range of CF regions. Coherent-reflection theory indicates that SFOAEs arise mostly from the peak region of the traveling wave, but several studies using farbasal suppressor tones claimed that SFOAE components originate many octaves basal of CF. We measured SFOAEs while perfusing guinea-pig cochleas from apex to base with salicylate or KCl solutions that reduced outer-hair-cell function and SFOAE amplification. Solution effects on inner hair cells reduced auditory-nerve compound action potentials (CAPs) and provided reference times for when solutions reached the SFOAE-frequency CF region. As solution flowed from apex to base, SFOAE reductions generally occurred later than CAP reductions and showed that the effects of cochlear amplification usually peaked ~½ octave basal of the CF region. For tones ≥2 kHz, cochlear amplification typically extended ~1.5 octaves basal of CF, and the data are consistent with coherent-reflection theory. SFOAE amplification did not extend to the basal end of the cochlea, even though reticular-lamina motion is amplified in this region, which indicates that reticular-lamina motion is not directly coupled to BM traveling waves. Previous reports of SFOAE-frequency residuals produced by suppressor frequencies far above the SFOAE frequency are most likely due to additional sources created by the suppressor. For some tones <2 kHz, SFOAE amplification extended two octaves apical of CF, which highlights that different vibratory motions produce SFOAEs and CAPs, and that the amplification region depends on the cochlear mode-of-motion considered. The concept that there is a single “cochlear-amplification region” needs to be revised.

Significance Statement The high sensitivity and frequency selectivity of mammalian hearing is attributed to cochlear amplification, but the spatial origin of cochlear amplification is poorly understood. Otoacoustic emissions (OAEs) are used for scientific and clinical purposes, e.g. to screen for hearing loss in well-baby nurseries, and can be measured over a wide range of frequencies in humans and animals. However, accurate interpretation of OAEs requires understanding cochlear amplification. Using a new experimental approach, we provide the most accurate assessment of the spatial pattern of cochlear amplification along the cochlear length, and are the first to show the amplification pattern in low-frequency cochlear regions important for speech. Our results will help in understanding the biophysical basis of cochlear amplification and in interpreting OAEs. 2

Introduction The sensitivity of mechanical responses in the mammalian cochlea is increased by the action of outer hair cells (OHCs) in a process called cochlear amplification. Amplification of basilar membrane (BM) traveling wave(s) is usually thought to take place in the region between a tone’s characteristic frequency (CF) place and approximately 1 octave basal of CF (1). The evidence for this comes from high-frequency regions of the cochlear base. In contrast, motion in lowfrequency cochlear regions is not like that in the high-frequency base (2-4). In low-frequency regions, cochlear amplification and its spatial extent are not understood. A noninvasive alternative to direct measurements of cochlear motion is the measurement of otoacoustic emissions (OAEs). OAEs receive cochlear amplification and can be measured from throughout most of the cochlear length in humans and laboratory animals. The most frequencyspecific OAE type is stimulus-frequency OAEs (SFOAEs). Coherent reflection theory indicates that SFOAEs arise mostly from the peak region of the traveling wave (5). However, several studies have purported to show that substantial SFOAE components originate many octaves basal of CF (6-10). Understanding SFOAEs and cochlear amplification require understanding how SFOAEs are measured. Measurements of SFOAEs are complicated by the overlap in both time and frequency of the evoking stimulus and the resulting emission. A common method for separating the emission from the stimulus is to calculate the difference between the response to a probe tone alone (P) and the response to a suppressor tone presented simultaneously with the probe tone (PS). The suppressor is thought to saturate OHC mechano-electric-transducer (MET) channels (11) thereby reducing the cochlear amplification that produced the SFOAE. When the suppressor frequency (Fs) is near the probe-tone frequency (Fp) and the suppressor level is ≥20 dB higher than the probe level, it is commonly assumed that the difference between the two measurements at the probe frequency (termed the “residual”) approximately equals the SFOAE produced by the probe alone:
 = − ≈ . Experiments with suppressor frequencies much higher than the probe frequency (Fs>>Fp) found significant residuals or wide suppression tuning curves (6,9,10), even for suppressor frequencies several octaves above Fp. These experiments were interpreted as showing that the SFOAE produced by the probe alone was a summation of sources from near the peak of the travelling wave and additional sources from more basal regions. It was further assumed that, like suppression at frequencies near Fp, the suppression of sources far basal of Fp resulted from a reduction of cochlear amplification. This interpretation goes against the view that cochlear amplification extends at most 1 octave basal of the Fp CF place.

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An alternative interpretation is that the Fs>>Fp residual arises because the suppressor, through cochlear nonlinearity, creates an additional backward traveling wave at frequency Fp that was not present when the probe was presented alone (12,13). Experiments done with Fs>>Fp established that intact cochlear amplification is needed in the suppressor-frequency region for a residual to be produced (10). However, these experiments did not distinguish between the hypotheses “the residual reveals that SFOAEs have far-basal sources” and “the residual contains additional sources introduced by the suppressor”. Both hypotheses require cochlear amplification in the suppressor-frequency region. One reason that previous experiments did not distinguish between the hypotheses is that their methods could not remove amplification in one frequency region without also affecting more basal regions. Here we address the issue of where the cochlear-amplification region is relative to the CF region using our recently-developed cochlear perfusion technique. We measured SFOAEs while perfusing the cochlea from apex to base at a preset rate with salicylate or KCl solutions that reduced cochlear-amplification sequentially from the lowest to highest CF regions. This technique provides, for the first time, a method that removes cochlear amplification and responses from one place along the cochlea without simultaneously affecting amplification or responses that originate basal to this place. The results allowed us to determine where along the cochlea cochlear-amplification was reduced and the distance between the cochlear-amplification reduction region and the CF region. This identifies where the probe-tone traveling wave was cochlear amplified relative to CF. The results also provide insight on where coherent-reflection theory fits the data and where it doesn’t.

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Methods Experimental Design We studied the origins of SFOAEs and the locations of SFOAE cochlear amplification (CA) by evoking SFOAEs with a probe tone while perfusing the cochlea from apex to base so that apical regions were affected before basal regions. The perfusion flow rate was adjusted to maintain a perfusion-front advance of 0.5 mm/min. We used salicylate or KCl solutions that interfered with OHC function so that CA was reduced sequentially from the lowest to highest CF regions. By also measuring the effects of these solutions on auditory-nerve (AN) compound action potentials (CAPs), we provided reference times for when the solutions reached the tone CF region (14). Both salicylate and KCl reduced CAPs when the fluid front reached the IHC region. KCl depolarizes IHCs and thus reduces CAPs when the solution reaches the IHCs. While salicylate’s effect on OHCs has been the most studied (e.g. ref (15)), salicylate also affects IHCs and can reduce CAPs (16,17). In a previous study, Lee et al. (14), we did cochlear perfusions with kainic acid (KA) solutions using the same cochlear perfusion technique used in the present study. KA blocks IHC-released transmitter from exciting AN fibers, but does not affect OHCs and CA (as shown by KA having no effect on OAEs) (14). Lee et al. (14) showed that, on average, KA reduced tone-evoked CAP responses by half at approximately the same time that an independent calculation of the fluid front location reached the tone CF location. Thus, the CAP reduction by KA provides a reference time for when the solutions reached the CF region. In our apex to base cochlear perfusions, KA, salicylate, and KCl solutions all reduced amplitudes of CAPs evoked from 40 dB SPL tone bursts at approximately the same time for a given frequency (Fig. 1). Thus, the perfusion time at which salicylate or KCl solutions reduced the CAP amplitudes identified the time at which the solution reached the IHCs that evoked the CAP, which for CAPs from lowlevel tones, is the region at the peak of the traveling wave, i.e. CF (14). KCl and salicylate reduce SFOAE amplitudes indirectly by reducing CA. Both salicylate and KCl reduce CA by affecting OHC function. Salicylate reduces CA by blocking prestin-based OHC motility (15) while KCl reduces CA by depolarizing the OHCs. These reductions in OHC function reduce the CA in the local region reached by the advancing fluid front in each oneminute measurement interval, but the effect is not just local because it is carried apically by the traveling wave. In each interval, how much a unit reduction of CA (e.g. 1 dB) reduces the SFOAE is a complex function of the location of the fluid front, how much amplification the traveling wave has accumulated at that location, and that a reduction at a given place will reduce the traveling wave amplitude and therefore will reduce BM motion at all more-apical locations (these more-apical locations will have already been reached by the fluid and had their CA removed, but their lowered-amplitude traveling waves can still evoke lowered-amplitude SFOAE components). Insight into this can be gained from Fig. 4 of Fisher et al. (18) where CA in various 5

regions has been reduced by inactivation of prestin. A 1 dB reduction of CA in the most apical part of the overall CA region will reduce the BM response in a narrow region but it will be a reduction of a high-amplitude response. In contrast, a 1 dB reduction of CA in the most basal part of the overall CA region will reduce a low-amplitude traveling wave, but this 1 dB reduction will be over a long cochlear region that extends to the apical end of the traveling wave. These two effects partly offset each other, but probably not completely, so that the amount that the SFOAE is decreased is an imprecise measure of the amount the local CA is reduced. In our experiment, the SFOAE reduction from the fluid flow in a one-minute interval indicates the total effect of the CA reduced during that one minute. This includes the competing effects of BM motion being reduced over a longer distance for more basal CA sources, and the CA reduction acting on a larger BM motion for more apical CA sources. These effects may provide uneven reporting of the local CA, but they do not produce a reduction of SFOAE amplitude outside of the CA region. The SFOAE decrease provides an approximate measure of the CA reduction as a function of place along the cochlea. Although the SFOAE reduction for a given fluid-front position is an uneven estimator of the CA from that region, we note that CA is often assessed by a more biased estimator: the nonlinear growth of BM motion. BM-response nonlinearity is caused by saturation (or just a slight nonlinearity) in local OHC MET channels which makes the local amplification nonlinear. The nonlinear response is carried by the BM traveling wave and accumulates nonlinearity going apically toward CF, even if the underlying incremental CA doesn’t change with place. Thus, the degree of nonlinearity is a biased indicator of CA. Furthermore, in the most basal CA region for a given frequency, OHC MET channels can provide CA for low-level signals while operating within their linear range. Such a region would be judged by its lack of nonlinearity to not have any CA. In contrast, our perfusion method would show such a region to be affected by CA reduction, and is therefore a better method than nonlinearity for showing the basal extent of CA. As an index of effective SFOAE reduction, we most often use the peak the SFOAE reduction rate. The SFOAE reduction rate shows the gain reduction produced by the fluid advance weighted by the effects described above. The uneven relationship between CA reduction and SFOAE reduction means that the location of the CA peak is not necessarily exactly at the SFOAE-reduction-rate peak. Another factor in interpreting our results is that due to KCl or salicylate diffusion at the fluid front, the advancing fluid produced a rapid, but not instantaneous, change in the local concentration. We estimate the time for the concentration at one location to rise from the threshold to the saturation of an effect on cochlear function to be 1-2 minutes (see Fig. 2B of Ref. 14). Diffusion would have spread out the times over which advancing solutions had effects, so that the spread of the KCl and salicylate effects shown in the figures is slightly slower than 6

would come from an instantaneous rise in concentration. This is true for effects on both CAPs and on SFOAEs. Animals Experiments used NIH-strain pigmented guinea pigs of either sex weighing 350 – 550g. Animals were initially sedated with intraperitoneal injection of 100 mg/kg Inactin hydrate (i.e., thiobutabarbital sodium) and then fur on the head and neck was shaved. A tracheotomy was performed for artificial ventilation with ~1-1.2% of isoflurane (to maintain anesthesia) in supplemental oxygen gas with respiration volume adjusted to maintain 5% end-tidal CO2. A pulse oximeter was used to monitor O2 saturation, expired CO2 level, and heart rate. Body temperature was maintained at 38°C with a DC-powered heating blanket and rectal thermometer system. The room was heated such that the area around the animal was ~30°C. The guinea pig head was secured with a bite bar, snout clamp, and ear bars. While in the supine position, a cannula was inserted into the left jugular vein and used to administer lactated Ringer solution every hour to maintain hydration (0.5 ml / hour). The right bulla was accessed ventrally by removing soft tissue and the jaw. The right ear canal was cut so that there were no acoustic leaks around the hollow ear bar. The left ear canal was not cut and a solid ear bar was inserted into the outer ear canal. Before data collection, vecuronium bromide (0.2 mg / kg) was administered through the jugular-vein cannula to prevent middle ear muscle contractions. Measurements were made with the guinea pig in a double-walled sound-treated room. At the conclusion of all experiments, animals were sacrificed by transcardial perfusion with fixative or with saturated KCl administered through the jugular-vein canula. All experimental protocols were approved by the Institutional Animal Care and Use Committee at Washington University in St. Louis (20150091, 20180133). Apex to base perfusions The mucosa layer on the bone of fourth cochlear turn was removed, dried with a cottonwrapped applicator, and covered with a thin layer of cyanoacrylate glue (#101, Permabond). A cup-shaped hydrophobic surface was made with silicone elastomer (Kwik-Cast, World Precision Instruments). Fluid in the cup-shaped surface could indicate leakage. A fenestra (~100-150 µm diameter) was made within the area of the cup-shaped elastomer using a House Oval Window Pick. The tip of a glass pipette (~50 µm diameter tip) was inserted into the fenestra and sealed with additional cyanoacrylate glue to prevent perilymph leakage. The cup-shaped surface gathered perilymph leakage that was wicked way when inserting the injection pipette and immediately before sealing the apical injection pipette into place. Solutions were perfused the through the apical fenestra, which is an established procedure in our lab (14,19-22). The perfused solution was 20 mM salicylate or 150 mM KCl in artificial perilymph. The 20 mM salicylate was prepared in artificial perilymph comprising NaCl (107.5 mM), KCl (3.5 mM), 7

NaHCO3 (25 mM), MgCl2 (1.2 mM), NaH2PO4 (0.75 mM), CaCl2 (1.3 mM) and C6H12O6 (glucose, 11 mM). The 150 mM KCl solution had NaCl (122.5 mM), KCl (150 mM), NaHCO3 (25 mM), MgCl2 (1.2 mM), NaH2PO4 (0.75 mM), CaCl2 (1.3 mM) and C6H12O6 (11 mM). The artificial-perilymph-only control contained NaCl (127.5 mM), KCl (3.5 mM), NaHCO3 (25 mM), MgCl2 (1.2 mM), NaH2PO4 (0.75 mM), CaCl2 (1.3 mM) and C6H12O6 (11 mM). Osmolarities of 20 mM salicylate and artificial perilymph were the same, but 150 mM KCl solution was hypertonic. Solution in the injection pipette was driven by a 50 or 100 µL gas-tight syringe (1710TLL, Hamilton Syringe) mounted on a programmatically-controlled ultra-pump (UMP3, World Precision Instruments). The pump rate varied at 1-minute intervals to achieve a constant flow of 0.5 mm/min along the systematically varying cross section of scala tympani (14,21). Perfused solutions exited at the base in the cochlear aqueduct. The total volume perfused was always greater than the ~6 µL guinea-pig scala tympani volume. The measurement system All cochlear function measurements were made from the right ear using a personal computer running custom MATLAB software (The MathWorks, Inc., Natick, MA) and the software utility Playrec. Stimuli were generated in MATLAB, digitized at 96 kHz, presented to a 24-bit sound card (RME: Fireface 802) and routed to an acoustic probe system (ER-10X, Etymōtic Research) that was coupled to a hollow ear bar (5 cm, 0.322 cm i.d.). Ear canal pressure measurements were made using the acoustic probe system connected to the 24-bit sound card. The ER-10X probe microphone was calibrated using a reference microphone (1/8” GRAS type 40P) and a long lossy copper tube (1.83 m; 0.635 cm i.d.) closed at one end. A sound source was placed in the open end. The probe and reference microphones were sealed into small holes 2.5 cm from the sound source, with the microphones located opposite each other and perpendicular to the long axis of the tube. We placed the ER-10X probe-microphone inlet and reference-microphone diaphragm flush with the wall of the tube. A train of click stimuli was played through the sound source, and the incident wave was measured simultaneously by the probe and reference microphones. The clicks were temporally spaced to allow internal reflections in the tube to decay into the noise floor before the next click was presented. Measurements were averaged and temporally windowed to include only the incident wave. From the results a transfer function of the ER-10X probe microphone relative to the reference microphone was calculated and used to achieve a flat probe microphone response from 0.1 to 34 kHz. To calibrate system output, the terminal end of the ear bar was coupled to a cavity having dimensions and volume similar to the cut guinea pig ear canal. The cavity was terminated by the reference microphone. Stimuli were played through the ER-10X probe loudspeakers into the ear bar and measured by the reference microphone. A transfer function was obtained and used to 8

calculate the stimulus levels in the experiments. In experiments, the ear bars were inserted to the cut ear canals with the tip extending into the bony portion and acted as stereotaxic ear positioners. The ER-10X was coupled to the hollow ear bar of the right ear; its sound sources presented the stimuli using the system transfer function described above and its microphone was used for recording otoacoustic emissions. CAPs were elicited by 40 dB SPL, 13.9 ms tone bursts (1.0 ms rise/fall times) alternating in polarity and interleaved with three 13.9 ms periods of silence, for a total of 69.5 ms and a repetition rate of 14.39/second. CAP measurements were made with an electrode placed in the round window niche, were band pass filtered at 0.1-3 kHz, and amplified 10,000 times (GRAS CP511, Astro-Med, Inc.). SFOAEs were measured using the double-evoked suppression method (23). In the more standard method that uses the difference between responses from the probe-tone alone vs. from the probe plus a suppressor tone, the suppressor part of the response is removed by filtering with a fast-Fourier-transform (FFT). In the double-evoked method, the suppressor portion is removed by including an additional stimulation period containing the suppressor alone (S), and computing the residual SFOAE from the three responses as:
 = + − ≈ . The double-evoked method reduces system distortions and cancels the effects of stimulus transients on the FFT. Probe tones were 40 dB SPL at Fp, and suppressor tones were 60 dB SPL at 50 Hz above Fp. The SFOAE stimulus series was a 250 ms probe tone, followed by a 250 ms suppressor tone, and finally 250 ms of probe and suppressor tones presented simultaneously. All had 10 ms rise/fall times. Noise floor measures were estimated as the standard error of the residual converted to dB SPL. During a cochlear perfusion, interleaved CAP and SFOAE measurements were made with one Fp per ear. Each minute, one block of measurements was saved to disk. There was ~3 seconds between measurement blocks. Each measurement block averaged the responses from two interleaved sets of CAP and SFOAE stimuli. A measurement set had 64 CAP responses and 24 SFOAE responses. Artifact rejection was performed post-hoc by sorting the response amplitudes into quartiles. Individual measurements falling outside of 1.5 times the interquartile range were rejected as outliers. We also removed portions of the recordings that contained bursts of noise from the fluid-perfusion pump. Preliminary tests were done shortly before the start of the cochlear perfusion to assess the general health of the ear. CAP thresholds were measured from 2 to 20 kHz in half-octave steps, and in some animals ANOW thresholds were determined at 300, 480, 720, and 1020 Hz (for ANOW measurements, see (24)). DPOAEs at 2f1-f2 were measured with primary tone frequency ratios f1/ f2= 1.22, L1 and L2 levels of 60 and 50 dB SPL, respectively, and f2 frequencies from 1 9

to 30 kHz in 2 kHz steps. All animals reported here had thresholds and DPOAE amplitudes within our normal range for guinea pigs. Fitting Procedure for CAP and SFOAE reductions during the cochlear perfusion CAP and SFOAE amplitudes, expressed in linear units (µV for CAPs and µPa for SFOAEs), were analyzed as functions of time relative to the perfusion start. CAP and SFOAE amplitudes start at positive values and decrease during the cochlear perfusion until they reach a steady state (e.g. the noise floor). Generally, the reduction curve was monotonic and smooth, but this did not always occur. When aberrant changes did occur, we analyzed the data over a shortened time window that excluded the aberrant change. This analysis window always included the times when, by visual inspection, the main reduction occurred. It also included periods of relative stability before and after the main reduction. The amplitude-versus-perfusion-time measurements in the analysis window were fit with a mathematical function chosen because it had enough flexibility to capture the underlying shape of the reductions (including asymmetry) with only two parameters. We used a regularizedincomplete-beta-function distribution ( (, )), defined for data points  laying in the interval [0, 1] as a function of shape parameters  and . In its original definition,  has a statistical meaning, but in the present application, it was used for its shape properties without reference to the original use. To use  , the data in the analysis window were appropriately scaled on the xand y-axes and the  function was flipped left-to-right along the x-axis (e.g., the solid lines in Fig. 2A show example  functions and their fit to the data points). For each set of scaled amplitude-vs-time data, a fitting procedure determined the optimal values (in the least-squares sense) of  and  using MATLAB’s function fminsearch, which finds the minimum of an unconstrained, multivariable function using the Nelder-Mead simplex method. The resulting  fit to the data was differentiated to yield its slope, and normalized so the area under the slope curve summed to 100% (e.g., the  two curves in Fig. 2A yielded the two slope curves in Fig. 2D). The slope curve is the rate of reduction as a percentage of the original amplitude. The peak of the rate-reduction curve is the time at which the reduction occurred most quickly. The intervals around the peak over which there were 68% or 95% reductions (the widths for ±1 SD and ±2 SD of a normal distribution) were used as metrics for where most, or almost all, of the reductions occurred. These were computed so as to give the shortest possible interval that included the desired percentage (which means that these intervals are not necessarily symmetric around the peak). Measurement times from start of perfusion, , were converted to distance (in mm) from the cochlear base, , using  =  −
 where
is the solution flow rate (0.5 mm/min), and  is the total distance of scala media (21.8 mm). Distances were converted to CF place using the guinea 10

pig place-frequency map of (25) using the equation CF = 10 ((%d-66.4)/-38.2), where %d is the percent distance from the base. The lower X-axes in Fig. 3 show conversions from perfusion time to CF location of the fluid front. Procedures for Fitting curves to Data Plots and Calculating Statistics To aid visual assessment, the plot of CAP reduction-rate-peak times versus frequency (Fig. 4A) was fitted with an exponential of the form y = a exp(bf), where f is frequency (kHz on a log10 scale). Solving for a and b used MATLAB’s Curve Fitting Toolbox with a robust algorithm (iterative bisquares re-weighting).

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Results CAP and SFOAE measurements were made while a salicylate or KCl solution was driven through the cochlea from the most apical part of scala tympani to the cochlear aqueduct in the base. This reduced cochlear function sequentially from low- to high-frequency cochlear places. Example CAP and SFOAE amplitudes as functions of time during a perfusion are shown in Fig. 2A. These measurements were fit by varying the two parameters of a single functional form (regularized-incomplete-beta-function distributions) as described in Methods (e.g. Fig. 2B, C). As indices of CAP and SFOAE reductions we used the slopes of the fits to the reduction-rate data (e.g. Fig. 2D). These slopes are the percentage reductions each minute of the CAP amplitude, or the SFOAE amplitude. They show the changes produced in each minute by the advance of the fluid front reducing CAP amplitude, or SFOAE amplification. In this example, the SFOAE reduction occurred more slowly than the CAP reduction, and the SFOAE reductionrate peak occurred after the CAP reduction-rate peak (Fig. 2D). The CAP reduction-rate peak is taken as an index of the time at which the solution reached the Fp CF region, i.e. the location of the probe-frequency traveling-wave peak (14) (see Methods for more detail). The SFOAE reduction-rate peak shows where the reduction of local CA has the maximum effect in reducing the SFOAE (CA effects on SFOAEs are weighted by basal CA affecting longer lengths of BM motion and apical CA acting on higher BM motion amplitudes – see Methods for more detail). The SFOAE reduction-rate region does not show where SFOAE wavelets originate from; it shows where reducing the CA reduces the SFOAE. The data in Figure 2 are consistent with the CA region being basal of the CF region. The reduction rates varied across ears, as shown by example data from four ears (Fig. 3). In all but one of these example ears (Fig. 3A), the amplification region was basal of the CF region. Measurements were made from twenty ears with Fp varied from 1 to 9 kHz. As Fp increased, the reduction of the responses occurred at later perfusion times, which is consistent with responses from higher frequencies being located more basally in the cochlea (Fig. 4). We did not find any statistically significant difference in these 20 ears between the effects of salicylate versus KCl (CAP: t(18)=-0.891, p=0.3849; SFOAE: t(18)=-0.601, p=0.556), or in the 17 ears tested with frequencies 2 kHz and higher, so for the analyses of this paragraph (which consider all of the data), salicylate and KCl data have been merged (they are not merged later). Averaged across ears, CAP reduction-rate peak times were significantly earlier than SFOAE reduction-rate peak times, with the CAP reduction-rate peak occurring an average of 2.87 minutes before the most rapid SFOAE reduction, (t(19)= 3.02, p= 0.007). In almost all cases, CAP responses were reduced to the 84% or 97.5% criterion-reduction values before SFOAE responses were reduced a comparable amount (Figs. 4D, 5A), and the differences were statistically significant by paired ttests (for 84% reduction: Ave. = 2.35 minutes, p=0.00025; for 97.5% reduction: Ave. = 2.48 minutes, p=0.00005). These statistics include the three points from frequencies below 2 kHz; if 12

these points are excluded, the statistical comparisons become even more significant. As Fp increased, the duration of the reductions (the time from 2.7% to 97.5% of the reductions) generally decreased (Fig. 5B). The reduction durations from 16% to 84% plotted versus Fp (not shown) looked similar to Fig. 5B. The relationship of the SFOAE-tone amplification region relative to the peak of the traveling wave is shown in Figure 6. In this figure, the times of SFOAE reduction-rate peaks and durations are expressed relative to the time of the CAP reduction-rate peak, with the time differences converted to octaves. The calculation of the octave distance used the flow rate of 0.5 mm/min and the cochlear frequency map of Tsuji and Liberman (25) (see Methods). It is convenient to consider the results in two frequency regions. For Fp≥2 kHz, the average time from the CAP reduction-rate peaks to the SFOAE reduction-rate peaks was equivalent to 0.573 octaves along the cochlea (which was statistically significant, t(16)= 4.204, p= 0.0007, SD= 0.561). The average offsets from the CAP reduction-rate peaks to the SFOAE reduction-rate 84% points and 97.5% points were 1.211 and 1.828 octaves, respectively (both were different from zero: p<0.0001). These averages are influenced by the point at 2 kHz that had an unusually long value (Fig. 6). More representative estimates of the octave distance from the CAP reduction-rate peaks to (a) the SFOAE reduction rate peaks, (b) the 84% decline points and (c) the 97.5% decline points, are the median values which are: 0.51, 1.16 and 1.62 octaves, respectively, for Fp≥2 kHz. For Fp<2 kHz, there are only three points and these have divergent values so an average would not be representative. However, we note that the average is negative, i.e. that the peak reduction of SFOAEs occurred before the peak reduction of CAPs. This surprising result will be considered in the Discussion. Despite the Fp<2 kHz SFOAE reductions starting and peaking before the CAP reductions, the SFOAE reductions ended approximately the same amount of time after the CAP reduction-rate peaks as did Fp≥2 kHz SFOAE reductions (for Fp<2 kHz, 84% and 97.5% SFOAE reductions averaged 0.82 and 2.00 octaves, respectively, basal of the CAP reduction-rate peak). After the perfusion front passed the SFOAE amplitude-reduction region, there was little or no further change in the residual from the ear-canal measurements. In some cases the perfusion removed all of the residual (e.g. Fig. 7C), but in others some residual remained (e.g. Fig. 7B). Since the remaining residual was close in amplitude to measurements done in an acoustic cavity, we attribute it to equipment nonlinearity. Whether or not there was a remainder, the advance of the fluid front would have reduced any cochlear amplifier gain in the region basal to the main SFOAE reduction. The lack of an effect in this region is consistent with there being no residual arising from traveling waves that are amplified within this basal region. This result indicates that Fp traveling waves were not receiving any CA within this region and that there are no suppressible SFOAE sources in this region. The remaining residuals were small compared to the 13

original SFOAEs so that calculating the SFOAE amplitude-reduction-rates with or without removing the remaining residuals had little influence on the data in Figs. 2-6.

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Discussion With a few exceptions, our data show that the greatest SFOAE reduction rate occurred after the greatest CAP reduction rate, and that SFOAE reductions ended after CAP reductions (Figs. 4B,C, 5A, 6). Both of these results are consistent with cochlear amplification extending basal of CF. The notable exceptions were at frequencies below 2 kHz where in 2 of 3 cases the SFOAE reduction started and peaked before the CAP reduction, which is a new and surprising finding that is discussed below. We found no significant difference between the effects of salicylate versus KCl when all of the data are considered or just the data above 2 kHz. Below 2 kHz the KCL and salicylate data diverge which allows for the possibility that they have different effects in low-CF regions, but with only 3 sets of data available, this possibility cannot be confirmed. By expressing the SFOAE reduction times relative to the CAP reduction-rate peak, we have removed much of the variation due to animal variations (e.g., different-size cochleas). If variation in the perfusion-effect times were caused by perfusion variations, such variations would get larger as perfusion time increased, i.e., at high frequencies. But, the data from highfrequency tones were generally more uniform than the data from lower-frequency tones (Figs. 4, 5B). In addition, this perfusion method has been used before and produced uniform results (14, 20-22). Also relevant is that SFOAE components are reflected by cochlear irregularities and these are expected to vary from one animal to the next. These considerations suggest that the variation in the data across experiments is mostly due to the cochlear frequency region sampled and to animal variations rather than to methodological variations. Cochlear amplification of reticular lamina motion in regions far basal of CF Reticular lamina (RL) motion in response to tones at frequencies lower than the local CF is amplified by active processes (26-28). Based on this, Siegel (29) hypothesized that when a suppressor is at a CF region several octaves basal of the Fp CF region, residuals at the Fp frequency (presumed to be SFOAE components) arise from suppressing the amplified motion of the RL. More recently, it has been shown that amplified RL motion from one tone can be suppressed by a second (suppressor) tone, but only if the suppressor tone produces motion at the measurement site and not if the suppressor-tone CF region is far basal of the measurement site (30). The implication is that the suppression of RL motion is local to the cochlear region near the suppressor-tone CF region, i.e. RL suppression occurs where the suppressor response deflects OHC stereocilia into a nonlinear range that reduces the local OHC amplification of RL motion. The observation that suppression of RL motion in a frequency region several octaves basal of the Fp CF region doesn’t couple to the Fp forward traveling wave implies that it also doesn’t couple to, or produce, backward traveling waves at the Fp frequency, i.e. it doesn’t contribute to producing Fp SFOAEs. That amplified RL motion doesn’t couple to traveling waves, suggests that even though there is amplified RL motion, the overall organ of Corti (OoC) motion doesn’t 15

produce a net displacement of scala-vestibule cochlear fluid. Perhaps upward RL motion is cancelled by an inward motion of the Hensen cells at the side of the OoC (31) or by tilting of the RL (32). Overall, the data indicate that, contrary to Siegel’s (29) hypothesis, residuals from suppressors with CFs several octaves basal of the Fp CF region do not arise from suppressing RL motion. An important observation is that after the solution front was past the SFOAE reduction region, there was no further residual change (Fig. 7). This is strong evidence that SFOAE CA regions are spatially limited and do not continue all the way to the basal end of the cochlea. It is thought that amplification of RL motion from tones at frequencies lower than the local CF continues for tones much lower than CF, and that amplification of RL responses to far-below CF tones extends throughout the cochlear base (30). Since the fluid front produces no further change in SFOAEs past the few-octaves wide SFOAE reduction region (Fig. 7), but the region basal of this is expected to have amplification of Fp RL motion, we conclude that amplification of RL motion in this region does not contribute to amplification of the Fp traveling wave. Thus, our data extend the conclusion of Dewey et al. (30) to guinea pigs and to previously-untested low frequencies in the frequency range important for speech. The Apical-Basal Transition A variety of data show that the pattern of cochlear motion changes around the middle of the cochlear length at an apical-basal transition region that varies in frequency across species. For the chinchilla, the transition frequency has been put at 4 kHz based on data from single auditorynerve fibers (33), but Charaziak and Siegel (9,10) found the Fp at which their chinchilla suppression data changed was lower (3 kHz). In guinea pigs, the transition frequency has been put at 3 kHz based on bends in SFOAE delay-versus-frequency plots (33), but for our guinea-pig data the transition appears to be lower (just below 2 kHz). It appears that the transition frequency depends on how it is measured, which may be because the changes along the cochlea are gradual and occur at different rates for different motions. Probe Tones Higher in Frequency than the Apical-Basal Transition For Fp ≥2 kHz, in all but one case, the reduction-rate peak occurred later for SFOAEs than for CAPs, and the average difference was equivalent to ~½ octave of cochlear distance (Fig. 6). From the median values for 84% and 97.5% SFOAE reductions (1.16 and 1.62 octaves basal of CF, respectively), we conclude that a single-number approximation for the basal extent of CA from CF is 1½ octaves, which would include approximately 90% of the effective CA for our median data from frequencies above the apical-basal transition frequency. Note that by using the term “effective CA” we include the weighting of CA effects on SFOAEs described in Experimental Design. 16

Measurements of nonlinear compressive growth in basal-turn BM motion have led to the conclusion that CA is greatest within ½ octave basal of CF and that amplification is restricted to the region within one octave basal of CF (e.g. 1, 34). Data consistent with this were found by Cody (35) who deduced the amplification region from the region where local damage reduced BM motion, by Fisher et al. (18), who determined CA locations from the regions where inactivation of prestin reduced BM motion, and by Fallah et al. (36) who determined the amplification region from BM nonlinearity and its relation to local extracellular voltages. Efferent inhibition of BM motion in response to clicks extended to frequencies one octave basal of CF, which implies that there was gain in this region (37). Our finding of peak effective SFOAE amplification ~½ octave basal of CF for Fp ≥2 kHz is consistent with these BM measurements. We found that the typical (i.e. median) effective SFOAE amplification region extended to approximately 1½ octaves basal of CF (Figs. 4B, 4D, 6), which is more than previous estimates. The previous data came from the cochlear base whereas our data extend much more apically. Auditory-nerve data indicate that tuning becomes wider in octaves going from base to apex, so CA can be expected to extend over a wider region in more apical regions than the 1 octave found in the basal regions typically measured. Finally, as noted in Experimental Design, the most basal edge of the CA region would not show a nonlinear BM response for low-level sounds because the OHC MET channels produce CA while operating within their linear range. In contrast, such a region would be revealed as producing CA by our perfusion method. Charaziak, Siegel and coworkers (8-10, 38) have published a large body of relevant data on both SFOAEs and transient otoacoustic emissions (TEOAEs) from chinchillas. They found that for frequencies above 3 kHz, suppression tuning curves of SFOAEs and CAPs were similar in general shape and in Q10’s, but SFOAE tuning was shifted to higher frequencies than CAP tuning. We found that the overall reduction times of SFOAEs were slightly longer than those of CAPs, but the difference was not statistically significant. In agreement with Charaziak, Siegel and coworkers (8-10, 38), we found that SFOAE reductions were at later times (i.e., arising from more basal, locations) than CAP reductions (Fig. 5A), and this effect was statistically significant. We attribute the CAP-to-SFOAE reduction time offset to the perfused solutions reducing CAPs by a direct effect on inner hair cells whereas the reduction of SFOAEs was from reducing the SFOAE gain which is located more basal than the traveling wave peak that excites CAPs. Probe Tones Lower in frequency than the Apical-Basal Transition: Effects basal of CF In most cases, the basal extent of the SFOAE-reduction region from the CF region was not much different for Fp <2 kHz than for Fp ≥2 kHz (Fig. 6). In contrast, with Fp’s below their apical-basal transition of 3 kHz, Charaziak and Siegel (8-10) found substantial residuals, or basal tuning-curve extensions, for suppressor frequencies several octaves above Fp. Although detailed 17

comparisons are difficult because of the differences in techniques and species, it appears that in many cases the residual amplitudes that Charaziak and Siegel (8-10) found for suppressors at various distances far basal of the Fp CF region are larger than would be expected from the amount of CA in these regions shown by the effective SFOAE reduction rates (Fig. 4D). To look in more detail at the differences between our data and Charaziak and Siegel’s, we first consider frequencies six times the probe frequency (6xFp), which is ~2.5 octaves above CF. This was the highest frequency tested in several relevant studies (6, 9, 10). Suppressors at a frequency of 6xFp sometimes produced Fp residuals with large amplitudes that were similar in amplitude to residuals from suppressors near the Fp frequency (Fig. 4 of (9); for TEOAEs see (38)). In our data, aside from one experiment, 6xFp frequency regions were reached by the fluid near the end of the SFOAE reduction when the SFOAE reduction rate had become very low (Fig. 6). These data show that there is usually very little amplification of the Fp response in the 6xFp region. Considering this, large residuals from suppressors at the 6xFp frequency are not likely to be due to suppression of SFOAE amplification in the 6xFp frequency region. The most economical hypothesis to explain large residuals for 6xFp suppressors is that these residuals come from new sources that are created by the presence of the suppressor tone, as suggested by the models of Talmadge et al. (12) and Shera et al. (13). Consistent with the hypothesis that these residuals come from new sources, as the suppressor frequency is changed these residuals show large phase changes reminiscent of the phase change in the Fp traveling wave (6,9,10). Note, the far-basal extension of a suppression tuning curve may be due to the far-basal suppressor tone producing new-source residual, which would require the “suppressor tone” level to be lowered until this residual was reduced below the tuning-curve criterion; this lowering of the suppressor tone produces a basal tuning-curve extension. From the previous paragraph, it seems likely that new-source residuals produced by far-basal suppressor tones account for much of the effects of far-basal suppressor tones found by Charaziak and Siegel (8-10). However, we did find one case (at 2 kHz, see Fig. 6) where the SFOAE reduction rate was high at the 6xFp frequency. There was no methodological reason we can identify for this result so we attribute it to animal variations (e.g. large cochlear irregularities basal of CF). If chinchillas had such variations more frequently than guinea pigs, that might account, at least partly, for the difference between Charaziak and Siegel’s data and our data. Charaziak and Siegel (9-10) found an abrupt change in behavior near 3 kHz: far-basal residuals or tuning-curve extensions were seen for Fp’s lower than 3 kHz, but not for Fp’s higher than 3 kHz. We didn’t find any corresponding abrupt change in the basal extent of the SFOAE reductions. Presumably, this difference is because Charaziak and Siegel used far-basal suppressor tones, but we did not. Charaziak and Siegel’s finding of a difference between Fp’s 18

above and below the 3 kHz apical-basal transition may be related to other findings that OoC motion is different above and below the apical-basal transition (4). Probe Tones Lower in frequency than the Apical-Basal Transition: Effects apical of CF A surprising finding from our probe tones at frequencies below the apical-basal transition is that in two out of three cases, the times of reduction-rate peaks, and the times of 2.5% and 16% reductions, were earlier for SFOAEs than for CAPs (Figs. 4B-C, 6). Figure 3A provides an example which shows that at times 0 to ~3 min, there was a substantial SFOAE reduction without any appreciable reduction of the CAP. This means that a substantial part of the SFOAE arose from OHC-based motion amplification that occurred apical of the region whose motion contributed to the CAP, and was more than 2 octaves apical of the CF region shown by the CAP reduction peak. Note that for our 40 dB SPL tone bursts, the CAP origin is close to the CF region (14). Previous work has also shown a different relationship of CA to CF at low CFs as compared to high CFs. When CA was reduced by furosemide injections, cat auditory-nerve fibers with CFs less than 1 kHz generally exhibited upward shifts in CF, while those with CFs above 1 kHz exhibited downward shifts in CF (39). Considering the three cases with Fp <2 kHz, a reduction of SFOAEs before CAPs was seen in the two salicylate perfusions but not in the KCl perfusion. This would be consistent with salicylate and KCl having different effects in the apex, even though there was no statisticallysignificant difference between their effects more basally. Since the basal data show considerable scatter, and there are only three points with Fp <2 kHz, we are unable to determine whether, or not, this difference is statistically significant; more data are needed. Although KCl’s effects on IHCs and OHCs comes from the same mechanism (cellular depolarization), salicylate’s effects on IHCs and OHCs come from different mechanisms. Salicylate’s effect on IHCs is the least studied, so it seems possible that the SFOAE-before-CAP reductions might come from an effect of salicylate on IHCs in the apex that is different from the effect in the base. However, Fig. 4A shows that for Fp <2 kHz, salicylate’s effect on CAPs is not substantially different from its effect for Fp ≥2 kHz. What is different for Fp <2 kHz is that the salicylate data show an unusually early start of the effect on SFOAEs (Figs. 3A, 4C, 6). These data imply that the Fp motion and CA responsible for SFOAEs extend apically of the CF region. Direct motion measurements in low-CF regions of unopened cochleas have been made for guinea pigs and gerbils (2-4). These measurements show low-pass-like tuning and that CA extends to frequencies both above and below the CF of the measurement site. In many of these measurements, the largest gain was for a tone at a frequency above the measurement-place CF, but since this was in the declining response region where the motion amplitudes were below the peak motion, its significance has received little consideration. With the usual conception of scaling symmetry (40), gain for a tone an octave above the CF of the motion measurement place 19

implies there is a similar gain for the CF-frequency tone at a location an octave more apical of the motion measurement place (note: scaling symmetry is only approximate in the apex (41)). Our data are consistent with motion measurements in low-CF regions (2-4) and with the hypothesis that an octave apical of the SFOAE-tone CF region there is gain for a motion that produces SFOAE components but this motion does not evoke CAPs, and this is the first gain to be reduced by the apex-to-base perfusion. How is it possible to have motion that produces SFOAEs without also exciting IHCs? CAP responses are produced by deflections of IHC stereocilia that are caused by a combination of radial shear between the RL and the tectorial membrane (TM) and fluid flow from changes in the RL-TM gap (42). Thus, the driver of CAP excitation is not the overall OoC motion at the RL (or BM), it is the relationship of RL motion to TM motion in both radial and transverse dimensions. That there is a discrepancy between auditory-nerve tuning and OoC mechanical tuning in the low-frequency apex has been previously pointed out and hypothesized to be from filtering that occurs after the mechanical drive to IHCs (cf. 2, 43). An alternate possibility is that RL and TM motions become more similar as frequency decreases below CF, thereby producing a low-pass filter in the IHC mechanical drive. With this hypothesis, apical of CF there is motion of structures that produce SFOAEs, but don’t produce CAPs because the RL and TM motions are the same. Whichever hypothesis is correct, our observation of SFOAE sources extending apical of the region that produces CAPs is consistent with the low-pass motion response found in the cochlear apex. Since the drive to IHCs is the difference of two motions, there must be more than one mode of OoC motion. Mechanical measurements provide direct evidence for the OoC exhibiting multiple modes of motion (28, 30, 31, 44). Auditory-nerve data show that these modes interact in complex ways throughout the cochlea to produce the drive to IHC stereocilia that leads to CAPs (reviewed by (45)). Although there is good evidence for multiple modes of OoC motion, we don’t know exactly what motion extends apical of CF and produces SFOAE components and how this and other modes interact to drive IHCs. The concept that there is a single “cochlear amplification region” needs to be revised. There is good evidence that the amplification region for the traveling wave is different than the amplification region for RL motion ((30), and see earlier in our Discussion). Wherever there are multiple modes of OoC motion (which is throughout the cochlea, e.g. (46)), it can be expected that each mode of motion couples in its own unique way to the forces of OHC motility and that the length of the cochlea over which this motility amplifies a particular mode of motion will differ for each mode. Thus, there can be different amplification regions for each mode. Coherent Reflection 20

Coherent reflection theory predicts that SFOAEs arise mostly from the peak region of the traveling wave (5). In contrast, the peak SFOAE reduction rate found by the current study was, on average, approximately ½ octave basal of CF, which, at first glance, might seem to go against coherent reflection theory. However, backward traveling waves are produced by reflections of the motion of the traveling wave, and the peak of motion is different from the peak of the traveling-wave amplification. Considering this, our data from tones above 2 kHz are consistent with coherent reflection theory. In this respect, the present data are consistent with data previously described by Lichtenhan et al. (47). Coherent-reflection theory originally assumed that there is a “broad-tall peak” of the traveling wave, as had been found in BM motion in the base of the cochlea (5). In the apex, OoC motion responses are low-pass, in contrast to the band-pass motion in the base. In the apex it is likely that there are other OoC modes of motion, in addition to the one that’s typically measured, as pointed out earlier. Because of the different motion patterns, it may be that coherent reflection theory does not apply in the apex in exactly the same way as it does in the base (as was concluded by (48)). Nonetheless, the basic concept of coherent reflection theory is likely to still apply, i.e. that to get a large Fp backward traveling wave toward the stapes, wavelets from a wide range of cochlear places have to add in phase (i.e. coherently). Further work is needed to determine how this concept works in the apex. At the very least, the region that generates the SFOAE in the apex seems likely to be wider than that in the base, and our data suggest that it includes regions apical of CF.

Conclusions 1. For tones ≥2 kHz, the effect of cochlear amplification on SFOAE amplitude usually peaks near ½ octave basal of the CF region. 2. For tones ≥2 kHz, the median values of our data indicate that the region that produces about 90% of cochlear amplification effects on SFOAEs extends to ~1.5 octaves basal of CF. 3. SFOAE amplification does not extend to the basal end of the cochlea, even though reticularlamina motion is amplified in this region. 4. SFOAE-frequency residuals from far-basal suppressors are most likely due to new sources created by the suppressor. 5. Apical of the Apical/Basal transition region, SFOAE amplification sometimes extended a few octaves apical of CF. 6. Our data support the hypothesis that the extent along the cochlea where there is motion amplification depends on the cochlear mode-of-motion being considered.

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Author Contributions JTL and JJG designed the research CL, JTL and SSG did the experiments SSG, JJG, and JTL analyzed the data JJG, SSG, and JTL wrote the manuscript.

Acknowledgments This research was supported by R01 DC014997 (JTL) from the National Institutes of Health, National Institute on Deafness and Other Communication Disorders. The authors declare that they have no conflicts of interest.

Figure Legends Figure 1. Times of auditory-nerve compound-action-potentials (CAPs) reduction-rate peaks for kainic acid, salicylate, and KCl solutions as functions of tone-burst frequency. All of these solutions reduced CAPs at approximately the same time for a given frequency. Figure 2. Example CAP and SFOAE amplitude reductions, and curves fit to these data (from animal DW168; probe frequency = 3.5 kHz; salicylate perfusion). A: CAP (red circles) and SFOAE (blue triangles) amplitudes versus time re. perfusion start. Amplitudes are linear measures normalized by their average values before the perfusion start. Dashed lines show noise floors. B and C: Amplitude-versustime data and the curves fitted to these data as described in Methods. D: Rate of reduction curves from the slopes of the curve fits in B and C, normalized so the area under each curve is 100. These curves show the percentage per minute of the original CAP amplitude, or the effect of SFOAE amplification, that originated from the cochlear place passed by the solution front at that time. At the top of panels B-D, symbols indicate the times of the reduction-rate peaks, and lines indicate perfusion times that encompass 68% reductions (thick lines), or 95% reductions (thin lines) around the peak. Figure 3. Example reduction-rate curves across a range of probe frequencies for CAPs (red thin lines) and SFOAEs (blue thick lines). Above the curves, symbols show the time points at which the most rapid reductions occurred and horizontal lines show the times over which 68% and 95% of the reduction occurred (thicker lines and thinner lines, respectively). The scale at bottom shows the CF region reached by the fluid-flow front at the time shown by the perfusion time scale above, calculated based on the fluidflow rate and the CF map of Tsuji and Liberman (25). Figure 4. CAP (red) and SFOAE (blue) peak reduction times and durations. In A and B, symbols show the times of peak reduction rates with open symbols for salicylate perfusions and filled symbols for KCl perfusions; the vertical lines show the duration times that encompass 68% (thick lines) and 95% (thin lines) rate-reductions. In C and D, the times of rate-reduction-duration beginnings (C) and ends (D) are shown by symbols (see keys in each panel). The curved line in A-D is a trend line fitted (see Methods) to the panel-A CAP peak-reduction points (the exponential fit had coefficients: a=13.61 (95% ci=10.39, 16.82) and b=0.7813 (95% ci=0.4573, 1.105). The bar in D shows a 1-octave time span and applies to all 22

panels. Multiple measurements at 2, 3, 3.5, 5 and 9 kHz have been plotted with slight frequency offsets to avoid overlap. Figure 5. A: SFOAE reduction end times versus CAP reduction end times. Symbols show the times for 84% or 97.5% reductions, for salicylate or KCl perfusions, as indicated in the key. Dashed line has unity slope. B: CAP and SFOAE reduction times (2.5% to 97%) versus the stimulus frequency. Lines are leastsquares fits to the data (CAP: thin red, SFOAE: thick blue). Figure 6. SFOAE reductions relative to CAP reduction-rate peaks. Symbols show the times of SFOAE reduction-rate peaks, with open symbols for salicylate perfusions and filled symbols for KCl perfusions. The vertical lines show the duration times that encompass 68% (thick lines) and 95% (thin lines) ratereductions (i.e. vertical lines are not error bars). The dashed line shows a fit to all of the points (see Methods) and the solid red line shows the average of the points at frequencies ≥2 kHz. Multiple measurements at 2, 3, 3.5, 5 and 9 kHz have been plotted with slight frequency offsets to avoid overlap. Figure 7. SFOAE-residuals as functions of perfusion time, for three experiments with probe frequencies (Fp) shown at top and experiment number underneath. The lines at bottom are the noise levels.

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