The auditory brainstem response in two lizard species.

Abstract

Although lizards have highly sensitive ears, it is difficult to condition them to sound, making standard psychophysical assays of hearing sensitivity impractical. This paper describes non-invasive measurements of the auditory brainstem response (ABR) in both Tokay geckos (Gekko gecko; nocturnal animals, known for their loud vocalizations) and the green anole (Anolis carolinensis, diurnal, non-vocal animals). Hearing sensitivity was measured in 5 geckos and 7 anoles. The lizards were sedated with isoflurane, and ABRs were measured at levels of 1 and 3% isoflurane. The typical ABR waveform in response to click stimulation showed one prominent and several smaller peaks occurring within 10 ms of the stimulus onset. ABRs to brief tone bursts revealed that geckos and anoles were most sensitive between 1.6-2 kHz and had similar hearing sensitivity up to about 5 kHz (thresholds typically 20-50 dB SPL). Above 5 kHz, however, anoles were more than 20 dB more sensitive than geckos and showed a wider range of sensitivity (1-7 kHz). Generally, thresholds from ABR audiograms were comparable to those of small birds. Best hearing sensitivity, however, extended over a larger frequency range in lizards than in most bird species.

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The auditory brainstem response in two lizard species
Elizabeth F. Brittan-Powella?
Department of Psychology, University of Maryland, College Park, Maryland 20742
Jakob Christensen-Dalsgaard
Department of Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
Yezhong Tang
Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, 610041, People’s Republic of China
Catherine Carr
Department of Biology, University of Maryland, College Park, Maryland 20742
Robert J. Dooling
Department of Psychology, University of Maryland, College Park, Maryland 20742
?Received 22 July 2009; revised 25 May 2010; accepted 2 June 2010?
Although lizards have highly sensitive ears, it is difficult to condition them to sound, making
standard psychophysical assays of hearing sensitivity impractical. This paper describes non-invasive
measurements of the auditory brainstem response ?ABR? in both Tokay geckos ?Gekko gecko;
nocturnal animals, known for their loud vocalizations? and the green anole ?Anolis carolinensis,
diurnal, non-vocal animals?. Hearing sensitivity was measured in 5 geckos and 7 anoles. The lizards
were sedated with isoflurane, andABRs were measured at levels of 1 and 3% isoflurane. The typical
ABR waveform in response to click stimulation showed one prominent and several smaller peaks
occurring within 10 ms of the stimulus onset. ABRs to brief tone bursts revealed that geckos and
anoles were most sensitive between 1.6–2 kHz and had similar hearing sensitivity up to about 5 kHz
?thresholds typically 20–50 dB SPL?. Above 5 kHz, however, anoles were more than 20 dB more
sensitive than geckos and showed a wider range of sensitivity ?1–7 kHz?. Generally, thresholds from
ABR audiograms were comparable to those of small birds. Best hearing sensitivity, however,
extended over a larger frequency range in lizards than in most bird species.
© 2010 Acoustical Society of America. ?DOI: 10.1121/1.3458813?
PACS number?s?: 43.64.Ri ?WPS?
Pages: 787–794
I. INTRODUCTION
The tympanic ear probably originated independently in
all major tetrapod groups ?Clack, 1997?. Lizards therefore
represent an independent experiment in tympanic hearing
and are highly interesting in comparative studies of ear evo-
lution in tetrapods. Most lizards have delicate eardrums and
sensitive ears ?Manley, 1990?, but the lack of behavioral as-
says of auditory sensitivity has made it difficult to compare
lizard hearing to other vertebrate groups. Relatively non-
invasive methods, such as cochlear microphonics ?reviewed
in Wever, 1978? and measurements of compound action po-
tentials recorded from the round window ?Werner et al.,
2008? have been used successfully, but the thresholds and
general frequency response of the ear are dependent on the
method used and the surgical preparation needed ?Manley,
1990; Werner et al., 2001. There is therefore a need for stan-
dardized, non-invasive measurements of auditory sensitivity.
The anatomy of the basilar papilla shows considerable
variation ?see reviews in Manley, 2000, 2002, 2004?. The
species chosen for this study, anoles and geckos, were se-
lected to reflect this variation. Anoles belong to the
Iguanidae and typically have short basilar papillae ?range
300–500 ?m? while the basilar papilla is both highly spe-
cialized and extended in Gekkonids ?range 1800–2100 ?m?
?Manley, 2002, Manley et al., 1999?. Furthermore, lizard pa-
pillae are divided into at least two distinct areas, one where
most hair cells respond best to frequencies below 1 kHz and
a second area ?or two in some species? that responds to
higher frequencies ?see Fig. 4.3 in Manley, 2000?. This high
frequency region is particularly well developed in gekkonids
?Manley et al., 1999; Manley, 2002?.
Despite the diversity in their papillae, all lizard species
share a similar hearing frequency range of about 0.1–5 kHz
?see Wever, 1978 and reviews in Manley, 2000; 2004?. In all
lizards investigated thus far, the auditory nerve fibers have
V-shaped tuning curves with lowest thresholds at 5 dB SPL
and show phase locking to low-frequency stimuli ?below ap-
proximately 1 kHz; Manley, 1981; Manley, 2000?. The re-
sponses of most auditory nerve fibers are primary-like and
characterized by robust onsets ?for review, see Manley,
2000?. Auditory nerve fibers project to the auditory brain-
stem ?Szpir et al., 1995, for review see Grothe et al., 2005?;
further neural processing along the auditory pathway has not
yet been investigated in detail ?Manley, 1981?.
The auditory brainstem response ?ABR? is as an effec-
tive tool to study hearing sensitivity as well as the function-
a?Author to whom correspondence should be addressed. Electronic mail:
bbrittanpowell@psyc.umd.edu.
J. Acoust. Soc. Am. 128 ?2?, August 2010© 2010 Acoustical Society of America 7870001-4966/2010/128?2?/787/8/$25.00
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ality of the auditory system, and is similar across most ver-
tebrate classes ?e.g., Corwin et al., 1982; Walsh et al., 1992?.
Most importantly, the ABR audiogram provides a good esti-
mate of the shape of the behavioral audiogram ?Borg and
Engström, 1983; Brittan-Powell et al., 2002; Stapells and
Oates, 1997; Wenstrup, 1984?. For most animals tested, the
ABR is unaffected by sleep and/or sedation ?Hall, 2007; Pic-
ton and Hillyard, 1974; but see Santarelli et al., 2003?. It is
manifested as a series of peaks occurring within the first 10
ms following stimulation and represents the progressive
propagation of neural activity through the ascending auditory
pathway.
This study compared hearing sensitivity of a vocal lizard
?Tokay gecko, Gekko gecko? to a non-vocal lizard ?anole,
Anolis carolinensis?. Tokay geckos are nocturnal Southeast
Asian animals. Gekkonoids are noted among lizards, both for
their ability to vocalize and for the complexity of their vocal
apparatus ?Blair, 1968; Bogert, 1960; Moore et al., 1991?.
Their vocalizations vary from quiet, insect-like chirrups to
loud clucks, barks, and whistles ?Marcellini, 1977; Seufer,
1991; Tang et al., 2001? and are distinguishable from those
of most other reptiles in that the sound emitted often has
tonal and harmonic qualities ?Brown, 1985; Steck, 1908;
Werner et al., 1978?. In contrast, the green anole is a diurnal,
non-vocal animal found in the southeastern United States.
We have used theABR to measure hearing sensitivity since it
is a simple, non-invasive measurement that allows compari-
sons of sensitivity and frequency response not only within
the group of lizards but also with other vertebrate groups. To
our knowledge, this is the first such study in lizards.
II. METHODS
A. Animal preparation
Geckos were placed in a walk-in sound-attenuating
chamber ?IAC? for all measurements. Closed, custom-made
sound systems were placed at the entrance of both ear canals,
containing commercial miniature earphones and miniature
microphones ?Knowles EM 3068?. After the sound systems
were sealed into the ear canal using Gold Velvet II ear im-
pression material ?Earmold and Research Laboratories,
Wichita, KS?, the sound systems were calibrated individually
at the start of each experiment, using built-in miniature mi-
crophones ?Knowles EM3068, Itasca, IL?.
B. Auditory brainstem response „ABR…
Hearing sensitivity was measured in 5 geckos and 7
anoles using ABRs. Anesthesia was induced in intubated ani-
mals by isoflurane inhalation via a chamber. Once the lizards
were anesthetized, a constant gas flow of carbogen mixed
with isoflurane at 1 ml/min was connected via a loose fitting
tube into the trachea ?Bennett, 1998?. Animals were sedated
with isoflurane in concentrations of 1% and 3%. At levels
below 1%, animals could be kept sedated but occasionally
took deep breaths and would respond to toe-pinch. When
changes to isoflurane levels were made, at least 30–60 min
were allowed before the next data collection began. Body
temperature was maintained at 27 °C by a heating blanket
wrapped around the animal. To measure ABRs, platinum
electrodes were inserted just under the skin at the vertex,
behind the stimulated ear and grounded at the other side of
the head. Responses to brief tone bursts, emitted through the
coupler sealed over the eardrum, were evoked at frequencies
between 0.2–10 kHz with intensity levels of 5 to 90 dB SPL.
The stimulus presentation, ABR acquisition, equipment
control, and data management are similar to those used in
previous bird and alligator studies ?Brittan-Powell et al.,
2002; Brittan-Powell et al., 2005; Higgs et al., 2002?.
Briefly, the system was coordinated using a Tucker-Davis
Technologies ?TDT; Gainesville, FL? System 3 modular
rack-mount system USB linked to a Dell laptop running TDT
‘BIOSIG’ and ‘SIGGEN’ software. Sound stimuli were gen-
erated using SIGGEN and fed through a RP2.1. The output
of the RP2.1 was connected to a programmable attenuator
?PA5?, which directly drove the speaker. Recording elec-
trodes were connected to the low-impedance Medusa Digital
Biological Amplifier System ?RA4L Headstage and RA16PA
PreAmp?, which added an additional 10–20? gain, and then
to the RA16BA ?Medusa Base station? via fiber optic cables.
All biological signals were notch filtered at 60 Hz during
data collection. After data collection, the signals were band-
pass filtered below 30 Hz and above 3000 Hz using BIOSIG.
As in previous studies, ABR thresholds were measured
in response to multiple-intensity stimulus trains consisting of
9 single frequency tone-bursts of increasing intensity and
presented at a rate of 2/s. Tone burst stimuli were 10 ms in
duration ?1 ms cos2rise/fall? with 20 ms ISI. Each ABR
represented the average of 200 alternating phase stimulus
presentations, sampled at 25 kHz for 285 ms following onset
of the stimulus. Each frequency-intensity combination was
replicated. Latency to wave 1 was calculated as the time ?ms?
to the first positive peak while the amplitude was measured
from baseline-to-peak for wave 1 ?see inset Fig. 2?B??. We
defined ABR threshold as the intensity 2.5 dB ?one-half step
in intensity? below the lowest stimulus level at which a re-
sponse could be visually detected on the trace between 1 and
6 ms, regardless of whether the peak in the waveform was
positive or negative ?Brittan-Powell and Dooling, 2004;
Brittan-Powell et al., 2005?.
The authors recognize that the brief stimuli and short
rise/fall times of the tone bursts used in this study were not
ideal for accurately determining thresholds for low frequency
sounds. However, increased rise/fall times and longer stimu-
lus durations affect the brainstem response morphology. In
humans, ABR peak latency was directly and positively cor-
related with stimulus rise time ?e.g., Hecox et al., 1976;
Kodera et al., 1977; Kodera et al., 1979? although Beattie
and Torre ?1997? found that increasing rise/fall time from 1
to 4 ms at 500 Hz had no effect on ABR threshold. Other
studies showed that increasing the duration of the tone re-
sulted in changes in waveform morphology, with decreased
amplitudes and offset responses in stimuli as short as 10 ms
?e.g., Kodera et al., 1977, see review in Hall, 2007?. Given
these constraints, we chose a similar methodology ?i.e., 5 or
10 ms tone trains with 1 ms rise/fall times? in order to com-
pare ABR thresholds for lizards with ABR thresholds from
small birds and alligators ?collected by the first author?.
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III. RESULTS
The typical ABR showed two to three prominent peaks
occurring within 4–6 ms of the stimulus onset ?Fig. 1?. Peak
1 of the ABR is the far-field representation of the negatively-
oriented peak of the compound action potential ?CAP N1? of
the auditory nerve, likely originating from along the tono-
topic gradient within the basilar papilla ?Jewett, 1970; Köppl
and Gleich, 2007?. Both geckos ?n=3? and anoles ?n=4?
showed a similar sequence of peaks in response to a click,
but geckos ?median: 27.5 dB pSPL? exhibited lower click
thresholds than anoles ?median 42.5 dB pSPL?.
As with all animals tested to date, ABR latency de-
creased ?Fig. 2?A?? and ABR amplitude increased ?Fig. 2?B??
with increasing sound pressure ?SPL? in both geckos and
anoles. There were no species differences in the latency
functions ?F?1,33?=1.6, p?0.05?. There were noticeable spe-
cies differences in the amplitude functions ?species; F?1,33?
=16.35, p?0.001?. In anoles, click-evoked ABRs gradually
increased in amplitude with increasing SPL, while geckos
showed large amplitudes and steeper slopes in response to
clicks. The slopes of the amplitude functions did not differ
?species x intensity F?6,33?=2.18, p?0.05?, but geckos
showed significantly larger amplitude responses in response
to suprathreshold levels ?55 dB and higher; F?2,33?=13.75,
p?0.0001? compared to anoles at the same SPL. When in-
tensity decreased below 55 dB pSPL, closer to threshold,
there were no differences in amplitude between the two spe-
cies ?F?5,33?=0.71, p?0.05?.
Exemplar tone evoked waveforms are shown for a single
gecko and anole ?Fig. 3?. Gecko ABRs showed better syn-
chrony, with more defined peaks, than anoles. Occasionally,
a small, low amplitude shoulder was evident on the rising
phase of wave 1; we assume this is a summating potential.
Some of the responses to low frequency ?e.g., 0.4 kHz?
stimulation revealed well-defined stimulus-locked peaks for
many of the geckos tested ?seen at 400 Hz in gecko in Fig. 3
but not in anole?. This frequency following response ?FFR?
was evoked at high intensity levels and masked later re-
sponse peaks at these levels. When intensity levels de-
creased, however, the amplitude of the FFR decreased rap-
idly. Also, since we used alternating phases during data
acquisition, note that the averaged FFR was about twice the
frequency of the stimulation. When tone stimulation in-
creased in frequency, sharper, more synchronized responses
were evoked, especially in the gecko. Note also that the
gecko response at 2 kHz was larger and better defined with
respect to the other frequency responses shown. Similarly,
enhanced responses were apparent in the anole ABR traces
but to a lesser degree.
Both species had U-shaped ABR-derived audiograms
and thresholds that were similar below 5 kHz ?Fig. 4?A??.
However, there were significant species ?F?1,105?=75.05 p
?0.0001? and species by frequency ?F?13,105?=11.91 p
?0.0001? interaction differences. Although geckos appeared
to have lower ABR thresholds than anoles, these were not
significant below 3 kHz ?with the exception of 1 kHz? ?post
hoc contrasts from species by frequency interaction showed
no significant differences at frequencies from 0.2–4 kHz,
F?8,105?=1.88, p?0.05?. Anoles were, however, about 20 dB
more sensitive than geckos above 5 kHz ?post hoc contrasts
from species by frequency interaction showed significant dif-
ferences from 5–10 kHz, F?6,105?=29.23, p?0.0001?. The
FIG. 1. Representative ABR click waveforms for a single gecko and anole
showing multiple waveform peaks within the first 4–5 ms following onset of
the stimulus.
FIG. 2. ?A? I/O functions ?medians and ranges? for peak 1 latency and ?B?
amplitude ?bottom? for click responses in 3 geckos ?triangle? and 4 anoles
?circle?. Inset: Schematic showing how latency and amplitude measurements
were taken.
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gecko showed well defined peaks in sensitivity around 0.6
and 1.6 kHz ??30 dB SPL? but a steeper high frequency
cutoff; whereas, the anole ABR-derived audiogram showed
extended sensitivity between 1–7 kHz.
The effects of varying isoflurane levels are shown in
Figs. 4?B? and 4?C?. Changing the level from 3% ?producing
full anesthesia? to 0.5%–1% ?animal is lightly anesthetized
or sedated?, produced no significant changes in sensitivity
?sedation level by frequency by individual F?52,84?=0.047 p
?0.05?.
ABR-derived audiograms for budgerigars, screech owls,
alligators and lizards show striking similarities but also dif-
ferences ?Fig. 5?. At low frequencies, both lizards and alli-
gators appeared to show increased sensitivity at and below 1
kHz when compared to the budgerigar and the screech owl
tested using the same stimuli, although sounds were deliv-
ered in free field for the birds. The differences between birds
and alligators were significant at 1 kHz ?F?4,43?=7.73, p
?0.0001?. Post-hoc Turkey’s HSD tests showed that birds
had significantly higher ABR thresholds at 1 kHz than anoles
or geckos at the 0.05 level of significance. All other compari-
sons were not significant. The opposite held for the mid-
frequency range ?between 2–4 kHz?, where geckos, anoles
and birds had similar thresholds, with alligators showing a
steep increase in ABR thresholds ?these were derived in air,
not water?. There were significant differences in thresholds at
2 kHz ?F?4,43?=6.47, p?0.0001?
=91.78, p?0.0001?. While post-hoc Tukey’s HSD showed
that the alligators had significantly higher thresholds than the
lizards and the birds at 2 kHz and 4 kHz, budgerigars and
geckos had similar thresholds, as did anoles and screech
owls, at 4 kHz. As frequency increased above 4 kHz, gecko
and budgerigar thresholds showed similar trajectories
?steeper increases in threshold?. ABR thresholds for anoles
were more similar to, but not as low as, the screech owl, an
and 4 kHz ?F?4,43?
FIG. 3. Representative ABR waveforms for a single gecko and anole, evoked by tones of 0.4, 0.8, 2, 4 and 5 kHz. Different scales for the highest stimulus
intensities are shown to the left of each averaged waveform. Arrows indicate estimated threshold. ?A? Response waveforms for 0.4 kHz at 70 dB reveal
well-defined frequency following response that masked later response peaks. Note that the use of alternating phases during data acquisition produces an
averaged FFR of about 2F. ?B? Response waveforms in the anole were smaller than those measured in the larger gecko. Note the large and sensitive response
to increasing stimulus frequency.
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auditory specialist. In both the anoles and screech owls, ABR
thresholds began to increase dramatically around 7 kHz. At 8
kHz,thresholds weresignificantly
=44.58, p?0.0001?, with post-hoc Turkey’s HSD tests
showing significantly lower thresholds in anoles compared to
all other species.
different
?F?4,24?
IV. DISCUSSION
A. Lizard hearing
The ABR proved a robust and useful method to evaluate
lizard hearing. We found no evidence that level of anesthesia
affected ABR thresholds in either species ?but see Dodd and
Capranica, 1992; Santarelli et al., 2003; Stronks et al., 2010?.
Overall, signals were relatively large in the two lizards, with
geckos showing better neural synchrony ?sharper peaks in
waveform? than anoles. The increased synchrony may be due
to geckos having three times as many auditory nerve fibers
as the anole ?Manley, 2000; Miller, 1985?. Interestingly, the
increased synchrony and larger amplitudes at higher sound
intensities did not translate to lower thresholds. As intensity
neared threshold levels, amplitude was similar in both spe-
cies. The audiograms of the two lizard species were similar
in frequency range and show similar peak sensitivity at 30
dB SPL. However, anoles were sensitive to a larger range of
frequencies ?thresholds ?30–35 dB SPL from 1–7 kHz?
and had significantly lower thresholds than geckos between
5–10 kHz. The increased high-frequency sensitivity in anoles
was consistent with experiments showing high frequency
spontaneous otoacoustic emissions among anolid lizards ?7.7
kHz, Manley and Gallo, 1997? and laser vibrometry mea-
surements showing extended high-frequency sensitivity of
the Anolis eardrum ?Christensen-Dalsgaard and Manley,
2008?. Unlike the anole, geckos showed distinct peaks in
sensitivity, one of which corresponds to the fundamental fre-
quency of their advertisement calls. The rattle ?mononote?
and the ge ko ?binote? call both show fundamental frequen-
cies around 500 Hz ?Tang et al., 2001?. Geckos also showed
increased sensitivity around 1.6 kHz which may correspond
to the second frequency peak at 1.3 kHz ?Brillet and Pail-
lette, 1991?. Lastly, low frequencies evoked FFR in high in-
tensity signals, especially in geckos, These responses were
twice the stimulation frequency, which is typical in studies
when opposite phases are added together ?see Aiken and Pic-
ton, 2008; Sohmer et al., 1977?.
We occasionally saw summating potentials that are gen-
FIG. 4. ABR audiograms for geckos and anoles and for two anesthetic
levels. Median thresholds ?and ranges? are shown for all data. ?A? ABR-
derived audiograms for anoles ?circles? and geckos ?triangles?. Note the
similar thresholds, except above 4 kHz, where anole audiograms were con-
sistently more sensitive. ?B? ABR audiograms for 3 geckos measured at 1%
and 3% isoflurane. ?C? ABR audiograms for 2 anoles measured at 1% and
3% isoflurane.
FIG. 5. ABR-derived audiograms for anoles ?closed circles? and geckos
?closed triangles? in comparison to budgerigars ?solid line, Brittan-Powell et
al., 2002?, screech owls ?dashed line, Brittan-Powell et al., 2005? and alli-
gators ?dotted gray line, Higgs et al., 2002?. Note all ABRs were measured
using the same equipment, except that the sound source for the bird and
alligator ABRs was free field while lizard sound stimuli were delivered
through earphones. Error bars for these average data are not shown for the
sake of clarity. Averaged data are shown for other studies, but median data
are shown for the anole and gecko for the sake of consistency.
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erated by summed inner-ear receptor potentials. Such sum-
mating potentials have been reported in round window re-
cordings in several species of lizards ?Johnstone and
Johnstone, 1969? and should be small in far field ABR re-
cordings. Although small, we consider summating potentials
here because they could contribute differentially to the ABR
and result in differences in thresholds in lizards with differ-
ent papilla organization. We think this unlikely because the
summating potentials, when observed, were much smaller
than wave 1 of the ABR. Furthermore, we defined ABR
threshold as the intensity 2.5 dB ?one-half step in intensity?
below the lowest stimulus level at which a response could be
visually detected on the trace between 1 and 6 ms, regardless
of waveform ?Brittan-Powell and Dooling, 2004; Brittan-
Powell et al., 2005?. Thus, although is possible that the
waveforms contain far field components of the summating
potential, given our definition of threshold, it is unlikely that
their small contribution affected the threshold estimations.
The comparable low best frequency ABR thresholds in
lizards, alligators and birds may reflect the similar organiza-
tion of their auditory systems ?Carr, 1992; Carr and Code,
2000; Gleich and Manley, 2000; Manley, 1970?. Birds, alli-
gators and lizards have unidirectional ?hair bundles oriented
in the same direction? populations of hair cells covered by a
tectorial membrane and similar auditory nerve projections
?Gleich and Manley, 2000; Leake, 1974?. Manley has pro-
posed that the primitive lepidosaur papilla supported unidi-
rectional hair cells covered with a tectorial membrane. Such
a papilla would only have responded to low sound frequen-
cies ?below about 1 kHz?. The evolution of additional unique
populations of high frequency hair cells in most lizards may
have increased lizard high frequency sensitivity ?Köppl and
Manley, 1990; Manley, 1990; Miller, 1980?.
B. Comparisons with earlier Tokay data
Comparison of previous data on audiograms in the
Tokay gecko obtained from single unit nerve recordings
?Eatock et al., 1981; Manley, 1972; Manley et al., 1999;
Sams-Dodd and Capranica, 1996?, cochlear microphonics
?CM? ?Werner and Wever, 1972?, and our ABRs, reveal dif-
ferences in audiogram shape ?Fig. 6?. The data fall into two
groups: one which shows a distinct low best frequency peak
and another which is more bimodal. The first group ?gray
lines? includes CM studies ?Werner and Wever, 1972? and a
single-unit study ?Sams-Dodd and Capranica, 1996? and
shows best sensitivity around 4–500 Hz and a relatively
monotonic decline of sensitivity from 600 Hz to 8 kHz. The
second group ?black lines? includes audiograms based on the
lowest thresholds of cochlear nucleus neurons ?Manley,
1972?, primary nerve fibers ?Eatock et al., 1981; Manley
et al., 1999? and our ABR data, and shows bimodal audio-
grams with best sensitivities at 400 Hz and 1–2 kHz. Each
method comes with its own set of limitations. CM measure-
ments may be biased toward low-frequency responses, where
the synchronization is greatest. Furthermore, Manley ?1990?
has pointed out that CMs from the bidirectional higher best
frequency population may largely cancel each other out be-
cause there are equally large populations with both orienta-
tions ?Manley, 1990; Köppl and Authier, 1995?.
Experimental data will further be influenced by the sur-
gical method used to access the auditory nerve, and both the
ventral surgical approach used by Sams-Dodd and Capranica
?1996? and the approaches used to record CM and compound
action potentials ?CAP? from the round window are consid-
erably more invasive than the present method. CAP and laser
interferometry measurements carried out in the same species
of geckos ?Eublepharis macularius and Oedura marmorata?
showed that the shape of the CAP audiograms ?very similar
to the CM audiogram in the Tokay? was not predicted well
by the tympanic membrane response. For example, the peak
response of the adult E. macularius velocity function was
approximately 2.2 kHz while the most sensitive CAP thresh-
old was at 0.7 kHz ?Werner et al., 2002?. It seems reason-
able, as suggested by Werner et al. ?2001?, that these differ-
ences are caused by the ventral surgical approach; surgical
fenestration of the ventral throat wall may cause artificial
enhancement of sensitivity at low frequencies and erratic re-
sponses at high frequencies. The role of the intact middle ear
cavity in high frequency hearing is also supported by recent
laser vibrometry studies and mathematical modeling of the
gecko middle ear that show a pronounced resonance of the
middle ear cavities ?Christensen-Dalsgaard and Manley,
2008?.
The bimodal audiogram is most consistent with laser
vibrometry studies of the Tokay gecko since the ABR peak
sensitivity at 2 kHz also reflects the maximal sensitivity of
the eardrumwithlaser
Dalsgaard and Manley, 2005; Werner et al., 2002?. At low
frequencies, the slope of the present ABR audiogram is
slightly shallower than for the previous physiological mea-
surements ?see Fig. 6?. Below 400 Hz, however, the very
measurements
?Christensen-
FIG. 6. Data showing the median gecko ABR audiogram ?black X’s? in
relation to cochlear microphonic data ?CM gray solid line; Werner and
Wever, 1972, Fig. 6?, lowest thresholds of cochlear nucleus cells ?black open
square: Manley, 1972, Fig. 2?, and VIIIthnerve data ?gray inverted triangle:
Sams-Dodd and Capranica, 1996, Fig. 2?A?; black triangle: Eatock et al.,
1981, Fig. 2? and lowest thresholds of auditory afferents ?black open circle:
Manley et al., 1999, Fig. 2?A??.
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brief tone pulse ?10 ms? is more broad-band than the nominal
frequency which may lead to frequency splatter and recruit-
ment of additional auditory responses from adjacent regions
of the papilla.
The 20 dB general difference in sensitivity between the
ABR audiogram and the single-unit audiograms is easier to
explain. As previously stated, ABR studies have shown that
frequency-dependent ABRs are good predictors of audio-
gram shape but not of absolute auditory sensitivity. ABR
thresholds are generally about 10–30 dB greater than behav-
ioral thresholds ?e.g., Brittan-Powell et al., 2002; Gorga
et al., 1988; but see Bullock et al., 1968; Popov and Supin,
1990?. The synchrony required for an ABR response may be
well above the threshold for behavioral sensation ?Hecox
et al., 1976; Szymanski et al., 1999?. In the barn owl, where
there is a large nerve data set, Köppl and Gleich ?2007?
compared measures of behavioral sensitivity with CAP, CM,
and single units and found very similar shapes of the CM and
CAP audiograms and relative thresholds of single auditory-
nerve fibers, although the CAP and CM thresholds were
much larger than the single unit or behavioral measures.
It should be noted that behavioral thresholds have not
been measured in most lizards ?or in other reptiles except
birds?, and although non-avian reptiles produce a variety of
sounds ranging from the rattling of snake tails to the bellows
of crocodilians ?see review Gans and Maderson, 1973?, in no
case has communication by sound been well documented
except in the gekkonid lizards. In the Tokay gecko, the domi-
nant frequencies of the call match the region of best sensi-
tivity in the ABR. Peak energy of vocalizations and maxi-
mum sensitivity of the audiogram usually match in birds and
frogs ?Ryan and Wilzcynski, 1988, Dooling et al., 2000?, and
the same relationship may hold for gekkonid lizards. How-
ever, the present results show that a non-vocal lizard ?anole?
has a more extended high frequency range and is as sensitive
as or more sensitive than the vocal gecko. This would sug-
gest that sound communication is not the prime function of
the exquisite auditory sensitivity in lizards.
ACKNOWLEDGMENTS
This work was supported in part by Grant No. P30
DC004664 to the University of Maryland Center for the Evo-
lutionary Biology of Hearing, by Grant No. DC00436 to
CEC from the National Institute of Deafness and Communi-
cative Disorders of the National Institutes of Health, and by
the Chinese Academy of Sciences Bairenjihua Grant No.
KSCX2-YW-R-077 to Y.T., and by grants from the Danish
Natural Science Research Council to J.C.-D.
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