J Comp Physiol A (2007) 193:601–612 DOI 10.1007/s00359-007-0215-0

O RI G I NAL PAPE R

Evoked cochlear potentials in the barn owl Christine Köppl · Otto Gleich

Received: 30 October 2006 / Revised: 22 January 2007 / Accepted: 3 February 2007 / Published online: 23 February 2007 © Springer-Verlag 2007

Abstract Gross electrical responses to tone bursts were measured in adult barn owls, using a single-ended wire electrode placed onto the round window. Cochlear microphonic (CM) and compound action potential (CAP) responses were evaluated separately. Both potentials were physiologically vulnerable. Selective abolishment of neural responses at high frequencies conWrmed that the CAP was of neural origin, while the CM remained unaVected. CAP latencies decreased with increasing stimulus frequency and CAP amplitudes were correlated with known variations in aVerent Wbre numbers from the diVerent papillar regions. This suggests a local origin of the CAP along the tonotopic gradient within the basilar papilla. The audiograms derived from CAP and CM threshold responses both showed a broad frequency region of optimal sensitivity, very similar to behavioural and single-unit data, but shifted upward in absolute sensitivity. CAP thresholds rose above 8 kHz, while CM responses showed unchanged sensitivity up to 10 kHz.

C. Köppl · O. Gleich Lehrstuhl für Zoologie, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany C. Köppl (&) Department of Physiology (F13), University of Sydney, NSW 2006 Sydney, Australia e-mail: [email protected] O. Gleich HNO-Klinik, Universität Regensburg, Franz-Josef Strauß Allee 11, 93042 Regensburg, Germany

Keywords Hearing · Auditory · Bird · Avian · Basilar papilla Abbreviations CAP Compound action potential CM Cochlear microphonic FFT Fast Fourier transform RMS Root mean square S-AMPA
-amino-3-hydroxy-5-methyl-4isoxazole propionic acid

Introduction The measurement of evoked potentials is Wrmly established as a fast and technically easy way of estimating sensitivity and other cochlear status parameters. Most commonly, a wire electrode is placed onto or near the round window. Depending on the stimuli and the Wlter settings for the recording, diVerent kinds of evoked potentials can be recorded which allow selective monitoring of hair-cell and neural function, respectively. For small laboratory mammals, the cochlear evoked potentials and the origins of the diVerent components are very well characterized (e.g. Dallos 1973; Sellick et al. 2003). In mammals, the principal components are: (1) the cochlear microphonic (CM), dominated by the alternating-current receptor potentials of the outer hair cells, (2) the summating potential, dominated by the direct-current receptor potentials of the inner hair cells and (3) the compound action potential (CAP), originating in the aVerent neurones and axons. For birds, this has not been established equally well, although round-window recordings are also a popular and useful technique (e.g. Chen et al. 1993; Gleich

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et al. 1995; Gummer et al. 1987; Patuzzi and Bull 1991; Saunders et al. 1973). With the present study, we aim to establish a normative data set for the adult barn owl, which can serve as a reference for studies of hearing development in the owl.

Materials and methods Twelve barn owls (10 Tyto alba guttata and 2 T.a. pratincola) were used. The experiments were performed in three separate series over several years, which, however, diVered only in some technical details. They will be referred to as experimental series 1, 2 and 3 below. Series 1 contributed only data on the CAP, in the later series 2 and 3, both the CAP and the CM were measured. Anaesthesia and surgery Owls were anaesthetised using combined intramuscular injections of ketamine hydrochloride and xylazine. Initial doses were 10 mg/kg ketamine and 3 mg/kg xylazine. Supplemental doses were approximately half of these, generally given at 45–60-min intervals. In experimental series 1, diazepam was occasionally added, at a dosage of 1.5 mg/kg. Heartbeat and breathing were continuously monitored via a diVerential recording between two Wne needles in the muscles of one leg and the contralateral wing. Body temperature was held constant at 39°C by a feedback-controlled heating blanket with the probe inserted into the bird’s cloaca. A metal pin Wrmly glued to the skull served to hold the owl’s head in a Wxed position. Recording potentials A silver-wire electrode, insulated except for a small bead melted at its tip, was placed onto the round window membrane of one ear. The electrode wire was either guided and held by a sheath of glass tubing or directly glued to the skull. The surgical approach diVered between the experimental series. In series 1, the skull was opened to primarily gain access to an area around the cerebellar Xocculus (for introducing glass microelectrodes (see Köppl et al. 1993) and the round window could not be directly seen during the experiment. However, landmarks allowed a reliable placement of the electrode by an experienced investigator on or near the round window. In experimental series 2 and 3, the skull was opened more posteriorly such that direct visualisation of the round window and columella was possible. In all cases, a grounded reference

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electrode (silver wire or Ag/AgCl pellet) was placed under the skin near the incisions made on the head. Electrode signals were ampliWed, band-pass Wltered and subsequently digitised and averaged. In experimental series 1, a Grass P15 ampliWer was used at 100£ or 1,000£ and the most restrictive bandpass Wlter settings used were 0.3–10 kHz. This signal was then fed to a custom-built computer interface and matching software digitising at a rate of 20 kHz and averaging 16 stimulus presentations. In series 2, a Grass P5 was used at 10,000£ ampliWcation and 0.1–30 kHz bandpass Wltering. In series 3, a Tucker–Davis technologies (TDT) DB4 ampliWer was used, at up to 100,000£ ampliWcation and 0.1 kHz high-pass Wltering. In both series 2 and 3, signals were then fed to a TDT AD1 analog-digital converter, connected via an O1 optical interface to an AP2 signal-processor interface in a personal computer. Thirty-two stimulus presentations were routinely averaged. Stimulus presentation The owls were placed in a sound-attenuating chamber for all measurements. Closed sound systems were used and individually calibrated for each owl. In experimental series 1, sound systems containing Beyer DT770 loundspeakers and Bruel & Kjaer 4133 microphones with probe tubes were inserted into both ear canals. In series 2 and 3, only one system containing a small earphone (Aiwa HP-V14 or HP-V541) and a miniature microphone (Knowles EM 3068 or FG3329) was inserted into the ear canal ipsilateral to the recording side, while the other ear remained open. Stimuli were tone pips of 10 ms (series 1) or 20 ms (series 2 and 3) duration, including 1 ms cosine-shaped rise and fall times, and delivered at rates of 4/s (series 1) or 5/s (series 2 and 3). Stimulus frequency was varied in kHzsteps, from 1 to 10 kHz, and at each frequency, responses to a series of levels were recorded, generally in 5 dB increments. In experimental series 2 and 3, step size was decreased to 3 dB near threshold and 500 Hz was added to the set of frequencies. For 500 Hz stimuli, the rise/fall times were extended to 2 ms. Stimuli were generated by a custom-built computer interface in experimental series 1 (20 kHz sample rate) and stimuli were presented with a free-running starting phase, which eVectively eliminated the CM-component of the response. In series 2, a TDT WG2 frequency synthesiser module was used, followed by an SW2 cosine switch and a PA4 attenuator. In series 3, stimuli were generated by a TDT AP2 digital signal processing interface (10 s sample rate), followed by an FT6 antialiasing Wlter and a PA4 attenuator. Stimuli for both

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series 2 and 3 had a Wxed starting phase and separate recordings were taken for CM and CAP, averaging equal numbers of stimulus presentations with opposite starting phase for the latter. Data analysis The CAP amplitudes were measured from the averaged recordings; in experimental series 2 and 3, an additional weighted running-average algorithm was applied to eliminate high-frequency noise on the recordings. Amplitudes were deWned as the diVerence between the Wrst negative peak N1 and the following most prominent positive peak. While the deWnition of N1 was straightforward (the minimum within the Wrst 5–6 ms after stimulus onset), we failed to Wnd an objective set of criteria that could be consistently applied to Wnd the following positive peak. However, an eVort was made to subjectively follow the same positive peak across one data set, i.e., across the diVerent sound levels presented at a particular frequency. Input-output functions were thus derived for each stimulus frequency. Response thresholds were determined visually, as the lowest level where a CAP was discernible if the next-higher level also showed a response and the next-lower did not. In addition, thresholds were determined from linear regression Wts through the initial segment of the curve (4–6 data points collected at the lowest stimulus levels), as the level eliciting a 5 V response. This method allowed us to complete some data sets where threshold had been narrowly missed, by extrapolation below the actually-measured SPL range. The criterion of 5 V was chosen because it corresponded, on average, to the visual deWnitions of threshold and lay clearly above the noise amplitude. In experimental series 2 and 3, noise in the recordings was routinely evaluated by determining the standard deviation of amplitude values within a 5 ms interval immediately preceeding the stimulus. The median standard deviation was §1.16 V, independent of stimulus frequency or level. Measures of CAP amplitude at Wxed sound levels (either in dB SPL or relative to the respective threshold) were obtained by linear interpolation between the two Xanking data points and never involved extrapolation. The CM amplitudes were measured from a fast Fourier transform (FFT) obtained simultaneously with the time-domain average (TDT SPEC or, later, TDT BioSig software). Peak values at the stimulus frequency were read oV and converted to V RMS, according to a calibration of the A/D hardware and associated software using sinusoidal signals of known amplitude. Input-output functions of sound level (in dB) versus

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CM amplitudes [in V RMS (root mean square)] were then derived. Thresholds were determined from exponential regression Wts through the initial segment of the curve (4–6 data points collected at the lowest stimulus levels), as the level eliciting a 1 V RMS response This value was chosen for comparability with previouslypublished studies. The noise Xoor of our recordings was below 1 V RMS at all frequencies except 500 Hz, where the median noise value (derived by averaging the two FFT bins that lay, respectively, 200 Hz below and above the stimulus frequency) fell at 1 V RMS. Histology One of the ears that had been treated with S-AMPA (
-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, Sigma A-0326; see below) was later investigated microscopically. After receiving an overdose of Napentobarbital, the owl was perfused transcardially with saline containing heparine, followed by 5% glutaraldehyde (EM-grade) in 0.1 M cacodylate buVer. The cochlea was isolated, postWxed in 1% OsO4 in buVer, dehydrated and embedded in araldite. Sections of 1 m thickness were cut, mounted on gelatine-coated slides and counterstained with toluidin blue.

Results Components of the evoked-potential waveform The CAP was generally a sharply deWned, negativelyoriented peak (N1) shortly after stimulus onset, followed by a more or less prominent positive deXection (P1). If stimuli were presented with invariable phase, the sinusoidal form of the CM was superimposed on this (example waveforms shown in Fig. 1). To support our interpretation of the waveform components, control experiments were carried out which selectively eliminated the neural cochlear component by destroying the aVerent synapses with S-AMPA (Reng et al. 2001). In two owls, after the routine measurements of CAP and CM across frequencies were completed, a small piece of dental gelatine sponge saturated with 10 mM S-AMPA in saline (0.9% NaCl) was placed onto the round window. After a minimum of 2.5 h, the sponge was removed and selected measurements were repeated for comparison with pre-exposure data. In one of these two owls, a sham experiment was also carried out on the contralateral ear, by placing a piece of sponge saturated only with saline on the round window for the same amount of time. In both S-AMPA treated ears,

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Fig. 1 Examples of recorded, averaged waveforms, from one individual ear. Responses to six diVerent stimuli are shown: 1, 5 and 8 kHz, each at a near-threshold level (d, e, f) and about 70 dB SPL (a, b, c). The time range shown is coincident with the stimuli. The high-level responses show superimposed the waveforms

obtained with constant stimulus phase (gray lines) and the waveforms with the CM averaged out by alternating opposite stimulus phases (black lines); the low-level responses (bottom row of panels) only show the latter. In addition, the deWnition of the N1 and P1 peaks is indicated by dashed lines

the CAP was severely aVected, while the CM did not change consistently (Fig. 2a, b). In the saline-treated ear, only slight changes of CAP and CM were observed (Fig. 2c). S-AMPA caused the CAP thresholds to rise to very high levels or become even undetectable for stimulation frequencies of 4–5 kHz and above. At these higher frequencies, S-AMPA abolished both the N1 peak and the following slower positive deXection, while leaving the CM almost unaVected (Fig. 2a, b). CAPs in response to lower frequencies were less severely aVected. Also, the eVect of S-AMPA appeared to be slower at low frequencies compared to high frequencies. The CAP in response to 7 kHz dropped rapidly after about 40 min of exposure, whereas at 3 kHz, the CAP was slowly and only mildly aVected after 90 min. It thus appeared as if the eVect was stabilizing and S-AMPA might never have fully reached the more apical regions of the 12 mmlong owl basilar papilla. Light-microscopical sections of one of the S-AMPA treated ears were evaluated at an apical papillar location approximately 18% from the apical end, corresponding to 1.6 kHz characteristic frequency (Köppl et al. 1993), at a midapical location (approx. 40%), corresponding to 4.6 kHz and a midbasal location (approx. 68%), corresponding to 8.3 kHz. Both the midbasal and midapical sections showed a severely distorted morphology with large vacuous spaces below the hair cells, where the aVerent nerve Wbres and synapses would normally be (Fig. 3b). At the apical location, the abnormalities were less severe and restricted

to the neural edge of the papilla (Fig. 3a). Such damage was not observed in ears of untreated owls, prepared with similar methods in our laboratory. The myelinated regions of neurones, outside the basilar papilla, appeared normal at all locations. The damage observed is consistent with that documented by Reng et al. (2001) after S-AMPA exposure in the pigeon, using more detailed transmission electron microscopy. The more extensive damage at midapical and midbasal locations compared with the apical, low-frequency location correlates with the extent of physiological eVects described above. In the second S-AMPA treated ear, selected CM responses were followed after injecting the animal with a lethal dose of pentobarbital. As soon as the Wrst eVects on breathing and heart rate were noticed, the CM began to decline and was reduced to the noise Xoor within a few minutes of breathing and heart failure (Fig. 4). After that, neither CAP nor CM potentials were detectable at any test frequency.

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Characteristics of the CAP The CAP data are reported from 12 barn owls. In two individuals, both ears were measured, with very similar results. In order to avoid mixing dependent and independent data sets, only one ear per animal was included in the Wnal data analysis shown here. Experimental series 1, 2 and 3 (see Materials and methods) will not be distinguished any further, as there were no apparent diVerences between their results.

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Fig. 2 The eVect of exposure to S-AMPA through the round window. Data from two individual ears are illustrated (row a and b of panels, respectively). Row c illustrates a sham experiment, exposing the round window to saline for an equivalent amount of time. The left graph in each row plots CM and CAP thresholds before and after treatment. Note the pronounced rise in CAP thresholds at high frequencies after exposure to S-AMPA, with only minor eVects on the CM. Exposure to saline alone had only minor eVects on both CAP and CM thresholds. On the right of each row, examples of waveforms are shown, for selected stimuli at 3 and 7 kHz, both before and after treatment

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N1 latencies decreased systematically with both increasing sound level and increasing frequency. A typical data set is shown in Fig. 5. To compare latencies across frequencies, Fig. 6 plots median latencies at 10 dB above threshold, which should minimize the eVects of varying threshold. At this level, latencies decreased from a median of 4.04 ms at 500 Hz to 2.61 ms at 9 and 10 kHz. When referred to a Wxed sound level, the extreme frequencies (500 Hz and 10 kHz), due to their higher thresholds, showed increased latencies (Fig. 5b). It should be pointed out that latencies in response to 500 Hz were probably also exaggerated by the increased stimulus rise time (2 vs. 1 ms at all other frequencies), used to reduce spectral distortion. CAP N1-P1 amplitudes increased with increasing sound level. A typical data set is shown in Fig. 7a; we refrained from averaging input-output functions across ears, because, due to individual calibration corrections applied after the experiments, the exact sound levels

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that were used diVered slightly for every ear and frequency. At most frequencies, the CAP amplitude grew steadily up to the highest sound levels tested. However, at 7 kHz and higher frequencies, input–output functions tended to Xatten at high sound levels in some ears (Fig. 7a). Correlated with that, a widening of the CAP peak and, at the highest levels, a double-peaked CAP was observed at those frequencies in all owls (e.g. Fig. 1c). In addition to the change with sound level, there was also a systematic change of CAP amplitude with frequency. Amplitudes showed a broad peak, centred around 6–7 kHz. This was similar whether they were taken at a constant level above threshold or referred to a constant, absolute level (Fig. 7b). The CAP shape was clearly asymmetrical in most cases, with the N1 showing a larger excursion than the P1, relative to the baseline before the stimulus. However, at the highest frequencies (7–10 kHz) there was a tendency for the N1:P1 ratio to be more symmetrical (compare Fig. 1a–c).

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Fig. 3 Morphology of a basilar papilla exposed to S-AMPA. a Cross-section at a location approximately 18% from the apical end, corresponding to 1.6 kHz characteristic frequency. The haircell areas indicated by frames are shown enlarged in the corresponding insets. b Cross-section at a location approximately 68% from the apical end, corresponding to 8.3 kHz characteristic frequency. Note the large, vacuous spaces (some of which are indicated by stars) where the aVerent Wbres and synapses are expected to be. The damage was much more severe at the basal location (b) compared with the apical location (a)

Individual CAP audiograms and median thresholds across all ears are shown in Fig. 8. CAP audiograms were broad with good sensitivity around 25 dB SPL over a wide range of frequencies. Only below 1 kHz and above 8 kHz did the sensitivity decrease markedly. The greatest variability between animals was observed at 8–10 kHz. There was no appreciable diVerence between subjective threshold evaluation and thresholds deWned by a set criterion (Fig. 8b).

Fig. 4 Change of evoked responses upon death of the owl. The thick black line plots the CM amplitude in response to 7 kHz, 88 dB SPL, followed after administration of a lethal anaesthetic overdose. The dashed lines labelled A, B and C point out the times of beginning changes in heartbeat, failure of spontaneous breathing and heart failure, respectively. Note the rapid decline of CM amplitude within the same time frame. The four insets illustrate response waveforms to 7 kHz, 88 dB SPL (top two traces) and 3 kHz, 81 dB SPL (bottom two traces), each before the overdose (left traces) and after heart failure (right traces). Note that this ear had already been exposed to S-AMPA and CAPamplitudes were thus already compromised before the lethal injection

500 Hz and lowest at 1–2 kHz, but showed no consistent trend over the remaining frequency range (Fig. 9b). When referred to a constant SPL, CM amplitudes showed a peak around 6–7 kHz (Fig. 9b). The CM thresholds varied little over the frequency range routinely evaluated (Fig. 10). The median thresholds fell between 40 and 50 dB SPL for all frequencies except 500 Hz, which lay a little higher at 55 dB SPL. In one animal, frequencies above 10 kHz were tested and showed a rise of thresholds with no measureable CM up to 67 dB SPL at 16 kHz (Fig. 10a).

Characteristics of the CM The CM amplitudes were characterized by a mostly linear growth with increasing sound level when plotted on double-logarithmic scales (examples in Fig. 9a). Deviations from this linearity, however, were often seen at low frequencies (up to 3 kHz), where the input-output functions tended to display a region of very slow amplitude growth at low sound levels (Fig. 9a). Also, there was a tendency for slower amplitude growth at high sound levels for the higher frequencies. Amplitude variations with frequency were less systematic for the CM than for the CAP. When taken at 20 dB above threshold, CM amplitudes were highest at

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Discussion Origin of the evoked responses Although a commonly used technique, the origins of gross evoked responses measured at the round window are poorly understood in birds. There is a more extensive literature about cochlear evoked responses in mammals, which provides important guidelines. However, due to the uncoiled shape and shorter length of the cochlear duct in birds, electrotonic spread and thus the relative contributions of diVerent cochlear

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Fig. 5 Change of CAP N1 latency with sound level and frequency, in one individual ear. a Latency as a function of sound level for six diVerent frequencies. Note the pronounced decline of latency with increasing level at all frequencies. b Latency as a function of frequency, for four diVerent sound levels, two absolute levels and two levels normalized to the respective thresholds. Note the general decline of latency with increasing frequency

locations to the summed response at the round window probably diVer between birds and mammals. Also, much less is known about the potentials produced by the tall and short hair cells of the avian basilar papilla in response to sound than about those generated by the mammalian inner and outer hair cells. We will Wrst discuss those aspects of our data that provide insight into the origins of the responses. Following that, unique properties of the responses in the barn owl will be highlighted. Separation of hair-cell and neural responses Control experiments veriWed that both the CM and the CAP component of the response were highly physiologically vulnerable. In addition, they could be diVerentially aVected by S-AMPA. This agent was

Fig. 6 Median CAP N1 latencies as a function of frequency, at a level 10 dB above the respective thresholds. Error bars indicate the range from the 25th to the 75th percentile. numbers above error bars give the sample size at each frequency

previously shown to damage the aVerent synapses on auditory hair cells in another bird, the pigeon, which resulted in severely elevated CAP thresholds (Reng et al. 2001). The same eVects were observed in the owl. Taken together, these data conWrm that the responses were of physiological origin and are basically attributable to hair cells (CM) and neural components (CAP), respectively. In contrast to the pigeon, CAPs in the owl were later and less severely aVected by S-AMPA at lower frequencies compared to those at higher frequencies, even after considerable waiting times. Because of its unusual length, the owl cochlea may behave more similar to mammals than to other birds in this respect. In the guinea pig, substances applied to the round window show a persistent concentration gradient, decreasing from base to apex, over similar time frames as used in our experiments (e.g. Salt and Ma 2001). CAP and otoacoustic emission measurements across frequencies suggest that such a concentration gradient exists even if the substances are delivered directly into perilymph (Chen et al. 2005). If something comparable happened with S-AMPA in our experiments, the concentration may have been too low apically to produce a measurable eVect. Snyder and Schreiner (1984) reported in the cat a neural component at the fundamental stimulus frequency which may be mistaken for CM under certain conditions. This component, termed auditory-nerve neurophonic, is presumed to be due to phase locking. Since phase locking in the auditory nerve is very prominent in the barn owl and extends to higher-than-usual frequencies (Köppl 1997c), it might be expected to contribute to the response identiWed as CM. There was no evidence for a consistent reduction of CM responses in the barn owl when the neural component was impaired by S-AMPA. Thus, there appeared to be no

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Fig. 7 a Input–output functions for CAP amplitude at six diVerent frequencies, from one individual ear. Note a tendency towards saturation at high sound levels at 7 and 9 kHz. b Median CAP amplitudes as a function of frequency, at a level 20 dB above the respective thresholds (closed symbols) and a Wxed level of 70 dB SPL (open symbols). Error bars indicate the range from the 25th to the 75th percentile, numbers above error bars give the sample size for 70 dB SPL, numbers below error bars for 20 dB re. threshold, at each frequency

signiWcant neurophonic component in the high-frequency responses identiWed as CM. However, we can be less certain at lower frequencies, where the elimination of neural responses was less complete. It is possible that neurophonic or other neural components contributed to the pronounced low-level nonlinearities observed in low-frequency CM responses, as shown in the gerbil (Henry 1995). The shape of our CAP responses basically agreed with previous descriptions of the CAP recorded under similar conditions in other bird species (Brittan-Powell et al. 2002; Chen et al. 1993; Patuzzi and Bull 1991; Sun et al. 2000; van Dijk 1973) and in mammals. The main characteristic is the sharp, negative N1 peak. Further peaks, however, were less distinct in our recordings and generally appear less distinct in birds than typically

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Fig. 8 a CAP threshold audiograms (threshold criterion: 5 V) from 12 owls. Values for each individual are joined by a line. b Median CAP thresholds as a function of frequency. The continuous line joins the median thresholds obtained with a criterion of 5 V CAP; error bars indicate the corresponding data range from the 25th to the 75th percentile; numbers near error bars give the sample size at each frequency. For comparison, the dashed line joins median CAP thresholds determined visually

seen in mammals (e.g. summaries in Dallos 1973; Sellick et al. 2003). Instead of sharply deWned P1 and N2, the waveforms often showed only a slight rippling or simply a smooth rise to a positive maximum. This probably represents a signiWcant DC component of neural origin in birds, as suggested by Patuzzi and Bull (1991) from scala-tympani recordings in the chicken, and as later conWrmed by Sun et al. (2000). The change in waveforms observed after S-AMPA treatment in the owl is consistent with this interpretation, as both N1 and P1 were eliminated at high frequencies (Fig. 2a, b). Local origin of the CAP The change of latencies with frequency was consistent with a localized origin of CAP responses along the tonotopically-organized basilar papilla. Latencies at lower

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Fig. 9 a Input–output functions for CM amplitude at six diVerent frequencies, from one individual ear. Note the distinctly nonlinear behaviour at low sound levels in response to 1 and 3 kHz. b Median CM amplitudes as a function of frequency, at a level 20 dB above the respective thresholds (closed symbols) and a Wxed level of 70 dB SPL (open symbols). Error bars indicate the range from the 25th to the 75th percentile; sample size was either 6 or 7 at the diVerent frequencies

frequencies are expected to be longer than at higher frequencies, both because of longer cochlear response delays and the longer conduction pathway along the axons to the round-window recording site. This general prediction was matched by our data. Also, the diVerence in CAP latency between frequencies was comparable to the range of click latencies of single auditory-nerve Wbres of diVerent characteristic frequency in the same species (Köppl 1997a). Some smaller deviations from the idealized expectation of a steady decrease in latency with increasing frequency are obvious in Figs. 5, 6. The disproportionate decrease from 0.5 to 1 kHz may be partly due to the longer stimulus rise time used for 0.5 kHz. The Xattening or even increase of latencies above 6 kHz matches qualitative predictions based on known changes in axon diameters across auditory aVerents in the owl, which peak at 4–5 m in

2

4

6

8

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Frequency (kHz)

Fig. 10 a CM threshold audiograms (threshold criterion: 1 V RMS) from seven owls. Values for each individual are joined by a line. In one ear, frequencies above 10 kHz were tested (note logarithmic frequency axis). b Median CM thresholds as a function of frequency. Error bars indicate the range from the 25th to the 75th percentile; numbers above error bars give the sample size at each frequency

the 7 kHz region and decrease to near 2 m towards the highest frequencies (Köppl 1997b). A second indication for a localized origin of the CAP was the change of CAP amplitude with frequency. CAP amplitude is determined by the number of synchronously Wring units (Dallos 1973). Thus, using pure-tone stimuli of similar temporal shape and given that the electrode has access to the whole auditory nerve, CAP amplitude should be proportional to the number of Wbres representing diVerent frequencies. The largest CAP amplitudes were obtained around 6–7 kHz and amplitudes fell oV towards both lower and higher frequencies. This matches the distribution of number of aVerent Wbres originating from the diVerent papillar locations in the barn owl (Köppl 1997b). The round window, our recording location, is situated at approximately the same level as the internal auditory meatus through which all papillar aVerents pass and thus also pass in close proximity to the electrode.

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Local origin of the CM

CAP CM single−unit best single-unit behav. (Dyson et al., 1998) behav. (Konishi, 1972)

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Characteristics of evoked potentials unique to the barn owl Compared to other birds, the barn owl shows extended sensitivity towards high frequencies (e.g. Fay 1988). This was well reXected in its CM and CAP audiograms, that showed a broad frequency range of good sensitivity. The shapes of the CM and CAP audiograms corresponded well to audiograms previously determined behaviourally (Dyson et al. 1998; Konishi 1973) and to the relative thresholds of single auditory-nerve Wbres across diVerent characteristic frequencies (Köppl 1997a; Fig. 11). Only at the very highest frequencies, 9 and 10 kHz, did the CAP thresholds rise faster than either behavioural or single-unit thresholds. This eVect was even more pronounced in an ABR audiogram reported for a single barn owl (Brittan-Powell et al. 2005) where threshold began to rise sharply above 6 kHz. It is possible that relatively few sensitive aVerent Wbers at these high frequencies could support a low behavioural threshold, but are not suYcient to generate a measurable evoked potential. In addition, the amplifying eVect of the owl’s facial disk, which is most pronounced at high frequencies (Coles and Guppy 1988), may be signiWcant. This was eliminated in our experiments through the use of closed sound systems.

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Threshold (dB SPL)

Recordings from a single electrode location such as the round window are a relatively crude method for sampling the CM. Even at low to moderate sound levels, where hair-cell responses will be spatially restricted, sites more distant to the electrode will be progressively attenuated. At higher sound levels, the recorded CM is likely to be a complex sum of hair-cell potentials from diVerent sites within the cochlea (Dallos 1973; Pickles 1982). In the absence of suYcient knowledge about many of the relevant parameters in birds, we cannot make any meaningful predictions about the relative amplitudes of CM across frequencies that can be compared to the actual data. The strongest evidence that the CM reXects local hair-cell activity from the corresponding tonotopic place along the avian papilla is an agreement between CM and CAP audiograms, as shown by Gleich et al. (1995) for the canary, by Patuzzi and Bull (1991) for the chicken and here for the owl (but see discussion below). In addition, in the chicken, the loss and subsequent recovery of CAP-thresholds after kanamycin treatment was paralleled by a similar frequency-speciWc loss and recovery of CM-thresholds (Chen et al. 1995).

60 50 40 30 20 10 0 -10 -20 -30 0.1

1

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Frequency (kHz)

Fig. 11 A comparison of diVerent measures of auditory sensitivity in the barn owl. Thick lines repeat the median CAP and CM audiograms of this study. Thin lines show two measures of singleunit sensitivity, derived from a sample of 335 auditory-nerve Wbres (own partly unpublished data): a second-order polynomial Wt (continuous line) and a (dashed) line joining the most sensitive thresholds. The gray lines show the behavioural audiograms of Konishi (1973) and Dyson et al. (1998)

Absolute thresholds for the CAP were close to, but consistently above average single-unit thresholds in the auditory nerve, and remained 20–30 dB above the most sensitive single-unit thresholds. Since the owl has a disproportionally large number of aVerent nerve Wbres (Köppl 1997b) and also shows less variation of singleunit threshold at any given characteristic frequency (Köppl 1997a), we had predicted that the CAP thresholds would be closer to the single-unit thresholds than in other birds. However, this was not the case. A similar diVerence of 20–30 dB between the two threshold measures was found in the chicken (see Salvi et al. 1992 for a direct comparison), while the diVerence appears to be larger for the pigeon (compare Sachs et al. 1974; Smolders et al. 1995; Gummer et al. 1987; Reng et al. 2001). Overall, birds appear to show a larger gap between single-unit thresholds and CAP thresholds than typical mammals (Dallos et al. 1978; Rajan et al. 1991). The CAP amplitudes, however, were clearly several times larger in the owl than in the chicken and canary, as measured with comparable methods and at comparable supra-threshold levels (Chen et al. 1993; Gleich et al. 1995; Sun et al. 2000). For example, at 4 kHz, CAP amplitudes were smallest in the chicken (<20 V at 60 dB SPL; Sun et al. 2000), intermediate in the canary (»30 V at 60 dB SPL; Gleich et al. 1995) and highest in the barn owl (>50 V at 60 dB SPL). This

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ranking reXects species-speciWc diVerences in aVerent Wbre numbers and regional innervation density (Köppl 1997b; Köppl et al. 2000) and conWrms a correlation of Wbre number and CAP amplitude also across bird species. At Wrst glance, the sloping saturation often observed in high-frequency input-output functions (Fig. 7a) might appear to be another unusual feature of the owl. However, similar behaviour has occasionally been shown in other birds (Gleich et al. 1995; Rebillard and Rubel 1981). In mammals, continued growth of the CAP at high level is attributed to recruitment of higher-frequency Wbres, responding within the tail region of their tuning curves (Özdamar and Dallos 1976). Given that avian tuning curves typically lack such tails, it is perhaps more surprising that CAP I/O curves often do continue to grow steeply at high levels in birds as well. Conceivably the typically large spread of single-unit thresholds at any one frequency in birds (reviewed in Gleich and Manley 2000) leads to continuous recruitment of additional responses up to high levels. Indeed, the barn owl shows an unsually tight distribution of single-unit thresholds among the birds studied (Köppl 1997a) which may contribute towards the saturating behaviour of its CAP at high frequencies. The CM amplitudes reported for birds vary widely (Gates et al. 1975; Gleich et al. 1995; Jorgensen 1977; Kim et al. 2006; Patuzzi and Bull 1991; Saunders et al. 1973; Sun et al. 2000) and our values for the barn owl fall within this range. Also, the high thresholds observed for the CM, relative to neural or behavioural measures (Fig. 11), appear to be typical for birds, although the threshold criteria employed by diVerent authors vary widely. However, the owl is unusual in that the CM persisted with nearly unchanged sensitivity and amplitude up to 10 kHz, the highest frequency routinely tested and the upper limit of single-unit characteristic frequencies (Köppl 1997a). In the chicken, CM thresholds rise signiWcantly before the respective neural characteristic-frequency limit, under comparable recording conditions, i.e. with the electrode on the round window and thus in relative proximity to the basal regions of the basilar papilla (Gates et al. 1975; Saunders et al. 1973). The same behaviour was observed in CM recordings obtained from within scala tympani in the chicken (Patuzzi and Bull 1991). In the canary, CM thresholds also rise above 2–3 kHz (Gleich et al. 1995). Although the neural frequency range of the canary is unknown, the upper limit for a small songbird is expected to be higher than that (Gleich et al. 2004). A possible reason for undiminished CM sensitivity at high frequencies in the owl may be a larger-than-usual number of hair cells due to the

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extended cochlear representation of high frequencies. The mapping constant increases continuously from the apex to the base of the owl papilla and surpasses 5 mm/ octave at the highest frequencies (Köppl et al. 1993). In less specialized birds, the mapping constants remain below 1 mm/octave (reviewed in Gleich et al. 2004). Thus, considerably more hair cells will respond to a given high-frequency stimulus in the owl. A second attractive possibility is the production of unusually large AC receptor potentials which may be a prerequisite to sustain neural phase locking up to 10 kHz in the owl (Köppl 1997c; Palmer and Russell 1986; Sullivan and Konishi 1984). Acknowledgments We are grateful to Hermann Wagner for his generous gift of two owls used in this study. GeoV Manley kindly commented on an earlier version of the manuscript. Supported by the Deutsche Forschungsgemeinschaft through the SFB 204, a Heisenberg fellowship and an individual grant (Sachbeihilfe Ko 1143/11) to CK. Experiments complied with the “Principles of animal care”, publication No. 86–23, revised 1985 of the National Institute of Health. Animal husbandry and experimental protocols were approved by the Regierung von Oberbayern (AZ 2112531-28/98).

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