The representation of conspecific sounds in the auditory brainstem of teleost fishes
Institute of Zoology, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria
* Author for correspondence (e-mail: a9403658{at}unet.univie.ac.at)
Accepted 28 March 2003
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Summary |
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Key words: auditory brainstem response, conspecific sound, temporal sound pattern, teleost, acoustic information, Platydoras costatus, Pimelodus pictus, Botia modesta, Trichopsis vittata, Lepomis gibbosus
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Introduction |
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Temporal sound patterns are also important in the communication system of
other animal groups such as frogs and insects. In the Pacific tree frog
Hyla regilla, two different call types that elicit distinct behavior
are discriminated based on interpulse intervals
(Rose and Brenowitz, 2002). In
many acoustically active insects, information about species identity is
primarily encoded in the temporal structure of the song (for a review, see
Römer, 1998
).
To date, only few data are available showing that the auditory system of
fishes is capable of resolving acoustic differences based on temporal
patterns. Marvit and Crawford
(2000) showed that weakly
electric fish of the genus Pollimyrus can distinguish interclick
intervals (ICIs) below 1 ms; moreover, their tone frequency and click-rate
detection thresholds indicate that natural sounds of Pollimyrus could
mediate species and individual recognition. Analyzing the auditory brainstem
response (ABRs) to double-click stimuli with varying click periods, Wysocki
and Ladich (2002
) showed that
the minimum click period resolvable by the auditory system was below 1.5 ms in
five hearing specialists. But how is a complex species-specific sound
consisting of several pulses varying in pulse periods and amplitudes
represented in the auditory system? Does this high resolution capability and
reliability of representation in the auditory pathway also hold true for a
series of repeated pulses, or do habituation or inhibition processes take
place?
In order to answer these questions, we recorded ABRs evoked by conspecific
sounds in different species. Bullock and Ridgway
(1972), using alert porpoises
(Tursiops truncatus), proved that ABRs to conspecific sounds can be
obtained. The first aim was to determine if and how complex conspecific sounds
are represented by the auditory system in fishes possessing different
sound-producing mechanisms and hearing abilities. In a second step, we
investigated which acoustical variables in communication sounds time,
frequency and/or amplitude are encoded in the auditory brainstem.
Finally, for the first time, we directly investigated the auditory sensitivity
to conspecific sounds. We investigated four representative hearing
specialists, which possess accessory hearing structures that enhance hearing
sensitivity and the frequency range perceived. In addition, we tested a
hearing generalist lacking accessory hearing structures, whose hearing range
is limited to the detection of lower frequency sounds (<1 kHz) of higher
intensities. Within the specialists, we investigated evoked responses of the
lined Raphael catfish Platydoras costatus (Doradidae) and
Pimelodus pictus (Pimelodidae) to conspecific broadband stridulatory
sounds and of P. pictus also to the low-frequency drumming sounds.
Furthermore, we tested orangefin loaches Botia modesta (Cobitidae),
which produce high-intensity, broadband knocking sounds emitted singly or in
series (Ladich, 1999
), and
croaking gouramis Trichopsis vittata (Belontiidae), which produce
broadband double-pulsed sounds
(Kratochvil, 1985
). Among
hearing generalists, we chose the pumpkinseed sunfish Lepomis
gibbosus, which produces broadband, rasping sounds with variable pulse
patterns and pulse durations (Ballantyne
and Colgan, 1978
).
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Materials and methods |
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Auditory brainstem response recordings
The ABR recording protocol used in this study followed that recently
described in Wysocki and Ladich
(2001,
2002
). Therefore, only a brief
summary of the basic technique is given here. During the experiments, the fish
were mildly immobilized with Flaxedil (gallamine triethiodide; Sigma Aldrich
Handels GmbH, Vienna, Austria). The dosage used was 1.31.6 µg
g-1 for P. costatus, 2.75.9 µg g-1
for P. pictus, 1.94.9 µg g-1 for L.
gibbosus, 0.30.5 µg g-1 for T. vittata and
3.37.5 µg g-1 for B. modesta. This dosage
allowed the fish to retain slight opercular movements during the experiments
but without significant myogenic noise to interfere with the recording. Test
subjects were secured in a plastic bowl (37 cm diameter, 8 cm water depth, 2
cm layer of fine sand) lined on the inside with acoustically absorbent
material (closed cell foam); in a previous study
(Wysocki and Ladich 2002
),
this proved to reduce resonances and reflections and thus to preserve the
temporal structure of broadband stimuli. Fish were positioned under water such
that the skin region between the nares and the medulla was 1 mm above the
surface; thus, the contacting points between skin and electrodes were not in
the water. A respiration pipette was inserted into the subject's mouth.
Respiration was achieved through a simple temperature-controlled
(24±1°C), gravity-fed water circulation system. The ABRs were
recorded using silver wire electrodes (0.25 mm diameter) pressed firmly
against the skin. The portion of the head above the water surface was covered
by a small piece of Kimwipes tissue paper to keep it moist and to ensure
proper contact during experiments. The recording electrode was placed in the
midline of the skull over the region of the medulla, and the reference
electrode was placed cranially between the nares. Shielded electrode leads
were attached to the differential input of an AC preamplifier (Grass P-55,
gain 100x, high-pass at 30 Hz, low-pass at 1 kHz). The plastic tub was
positioned on an air table (TMC Micro-g 63-540; Technical Manufacturing
Corporation, Peabody, MA, USA) that rested on a vibration-isolated concrete
plate. The entire set-up was enclosed in a walk-in soundproof room, which was
constructed as a Faraday cage (interior dimensions: 3.2 mx3.2
mx2.4 m).
Sound stimuli were presented and ABR waveform recorded using a modular rack-mount system [Tucker-Davis Technologies (TDT), Gainesville, FL, USA] controlled by an optically linked Pentium PC containing a TDT digital-processing board and running TDT BioSig 3.2 software.
Sounds presented
Sound stimuli for hearing specialists were chosen among previously recorded
(Ladich, 1998,
1999
; Wysocki and Ladich,
2001
,
2002
) representative
conspecific sounds. All stimuli were complete sounds as emitted by the fish,
except the drumming sound of P. pictus, which was shortened by
approximately half. For T. vittata, a sound consisting of three pairs
of pulses was taken in order to avoid a measuring time that was too long.
Among B. modesta, which emit single or a series of knocks with a long
pulse period, we chose one sound consisting of two and another of three
knocks. For the hearing generalist L. gibbosus, a sound consisting of
four pulses was chosen from field recordings provided by Kurt Osterwald. A
second sound stimulus was created by eliminating the second and third pulses
of the four-pulsed test stimulus using CoolEdit 2000 (Syntrillium Software
Corporation, Phoenix, AZ, USA).
In addition, control tests using heterospecific sounds were performed in order to test whether the responses observed are specific to conspecific sounds. Sounds of L. gibbosus, T. vittata and B. modesta (three-pulsed sound) were presented to four individuals of P. pictus, and sounds of T. vittata, B. modesta (three-pulsed sound) and P. pictus (stridulation sound) were presented to four individuals of L. gibbosus.
All sound wave files were imported into TDT SigGen 3.2 software and fed
through a DA1 digitalanalog converter, a PA4 programmable attenuator
and a power amplifier (Denon PMA 715R, Alesis RA300). A dual-cone speaker
(Tannoy System 600, frequency response 50 Hz15 kHz±3 dB),
mounted 1 m in the air above test subjects, was used to present the stimuli
during testing. Stimuli were presented to the animals at repetition rates of
210 per second according to the length of the stimulus. A hydrophone
(Brüel & Kjaer 8101; Nürum, Denmark; frequency range, 1
Hz80 kHz±2 dB; voltage sensitivity, -184 dB re 1 V
µPa-1) was placed close to the right side of the animals (2 cm
away) in order to control for stimulus characteristics [such as sound pressure
level (SPL), sound spectrum and temporal structure]. SPLs of sound stimuli
were measured by a Brüel & Kjaer 2238 Mediator, Brüel &
Kjaer 2804 power supply and Brüel & Kjaer hydrophone 8101 (time
weighting, RMS Fast; frequency weighting, linear between 20 Hz and 20 kHz).
For each test condition, stimuli were presented at opposite polarities and the
ABRs to the two stimulus phases were averaged by the BioSig software in order
to eliminate stimulus artefacts. In order to create a 180° phase-shifted
stimulus, a copy of each original signal was inverted by 180° using
CoolEdit 2000 (for illustration, see Fig.
1). Each response waveform represents an average of
14002000 stimulus presentations over an analysis window of 50400
ms using a sampling rate of 20 kHz. Sound pressure levels of stimuli were
reduced in 4 dB steps until the ABR waveform disappeared. The lowest SPL for
which a repeatable ABR trace to any of the presented sound pulses could be
obtained, as determined by overlaying replicate traces, was considered the
threshold (Kenyon et al.,
1998).
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ABR waveform analysis and statistics
The following characteristics of sounds and brainstem responses were
analyzed: number, latencies, amplitude and frequency content of responses.
Latencies of the response were defined as the time interval between the onset of the sound stimulus and the first negative peak of the ABR waveform. Amplitudes were measured from the first negative peak to the most constant positive peak in each species. Wherever a given waveform could be related to a corresponding pulse of the sound stimulus, latencies and amplitudes of the ABRs and the sound pulses 20 dB above the mean hearing threshold of each particular species to this sound were compared by two-tailed correlations [Pearson's correlation coefficient was used when a previous KolmogorovSmirnov test showed that data/sound stimulus characteristics (temporal structure, peak amplitudes) were normally distributed]. All statistical tests were run using SPSS version 10.0.
In order to analyze whether the main frequency content of sounds was
represented within ABRs, fast Fourier transformations (FFTs) of sound stimulus
waveforms and corresponding ABR waveforms were performed using S_Tools, the
Integrated Workstation for Acoustics, Speech and Signal Processing developed
by the Research Laboratory of Acoustics at the Austrian Academy of Sciences.
Only sound stimuli with a main energy content below 1 kHz were analyzed
because of the filter settings of the electrode preamplifier (low-pass at 1
kHz) and because human ABR audiometry has revealed that there is little
spectral ABR energy at frequencies above 2 kHz. Evoked-potential studies on
mammals showed that certain frequency contents of sounds, such as formants of
vowels (Krishnan, 2002), are
reflected by scalp recorded auditory potentials; this is attributed to
phase-locked activity in populations of neural elements within the brainstem.
Spectral peaks were compared between the FFT spectrum of the stimulus and the
corresponding mean ABR spectrum generated using individual spectral data of
each species. Spectral analyses included the whole drumming sound stimulus of
P. pictus, the first pulse of the two-pulsed sound and the third
pulse of the three-pulsed sound of B. modesta and their corresponding
ABRs. Only a single pulse of each sound of B. modesta was chosen,
because the intervals between pulses were so long that an averaged spectrum
would have contained a large amount of background noise, possibly influencing
the spectrum. For the same reason, spectral analysis in L. gibbosus
concentrated on the first pulse of the sound in which pulses 2 and 3 were
omitted; this was also the only pulse to which a separate ABR trace could be
attributed and it would have been difficult to interpret a spectrum of a
waveform consisting of several superimposed ABR traces.
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Results |
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In B. modesta, the latencies to the two tested stimuli differed from one another considerably: the three short pulses (pulse periods 145.9 ms and 170.9 ms) evoked a fast response, whereas the sounds consisting of two longer pulses (pulse period 125.4 ms) caused a longer latency in ABR waveforms (Fig. 2B; Table 1).
The sound stimulus for T. vittata consisted of three pairs of pulses (Fig. 2C), each pair representing the alternating plucking of two enhanced tendons over the bony basis of the rays of two pectoral fins. Single-pulse periods within a pair of pulses were 6.3 ms, 10.9 ms and 6.9 ms and double-pulse periods were 47.5 ms and 45 ms. ABR waves to the separate stimulus pulses of croaking gouramis had the shortest overall latencies of all species (Table 1). Similar to both catfishes, ABR latencies were also negatively correlated to stimulus pulse amplitudes (r=0.828, P<0.05).
In contrast to hearing specialists, specific ABR waves in L.
gibbosus could not be attributed to the separate pulses of the complete
conspecific sound stimulus (Fig.
2C) consisting of four pulses (pulse periods 25.6 ms, 29.4 ms and
33.3 ms). When pulses 2 and 3 were omitted from the sound stimulus, a response
to the first pulse lasting approximately 51.7 ms could be differentiated from
the response to the second pulse 88.3 ms apart
(Fig. 3). The response to the
second pulse was also longer than those of the hearing specialists, but it
exceeded the duration of our 120 ms time window and was therefore not
measurable. The first discernible ABR deflection started about 1.8 ms after
onset of the first stimulus pulse of the sounds. For the modified sound
(pulses 2 and 3 omitted, pulse period 88.3 ms), the mean latency to pulse 4
was 3.2 ms (Table 1). The lack
of four separate responses to the unmodified sounds does not necessarily mean
that pulses 2 and 3 are not represented in the auditory brainstem of the fish:
the responses are quite long and could simply be superimposed. In order to
test this for at least pulse 2, a point-to-point subtraction procedure (for
details see Wysocki and Ladich,
2002) was performed: the response to the modified sound was
subtracted from the response to the complete sound. After this subtraction, a
response to pulse 2 was discernible with a mean latency intermediate between
that of pulses 1 and 4 (Fig. 3;
Table 1), to which a response
to pulse 3 could be superimposed.
Representation of amplitude patterns within ABRs
Correlating the amplitudes of conspecific sound pulses to the amplitude of
the corresponding ABR waves revealed differences between species: a
significant correlation in amplitude was measured in P. costatus
(r=0.570, P<0.001;
Fig. 4A) and in B.
modesta for both sound stimuli (r=0.705, P<0.001 for
the sound consisting of two longer pulses, and r=0.799,
P<0.001 for the sound consisting of three short pulses). In T.
vittata, the correlation was close to significance (r=0.314,
P=0.062; Fig. 4B). In
P. pictus, neither amplitudes of the stridulation sound
(r=0.251, P=0.082) nor of the drumming sound
(r=0.146, P=0.368) were correlated significantly to
the amplitudes of the corresponding ABR waves. Because of the lack of clear,
short, separated ABRs to each stimulus pulse, no such correlation could be
made in L. gibbosus.
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When approaching the hearing thresholds, brainwaves evoked by the less-intensive stimulus pulses disappeared: at threshold, an individually different number of the more-intensive sound pulses usually at mid-stridulation elicited a response in the two catfishes. For the drumming sound of P. pictus, the response to the first pulse always persisted at hearing threshold, while a variable number of the subsequent pulses disappeared in the different individuals. In T. vittata, the first pulse of the first double pulse, which had a considerably lower amplitude than the rest of the stimulus (Fig. 2C), evoked no ABR at hearing threshold. In B. modesta, at hearing threshold, the pulse with the highest amplitude (pulse 1 of the low-frequency sound, pulse 3 of the broadband sound) was always last represented in the ABR. In L. gibbosus, it was not possible to correlate a particular part of the ABR to a specific stimulus pulse for the unmodified sound. The last ABR waves persisted in the middle of the stimulus. For the modified sounds consisting of two pulses, both pulses were represented by the ABR waves at hearing threshold.
Representation of frequency contents
The drumming sound stimulus of P. pictus showed a harmonic
structure with spectral peaks (corresponding to the four harmonics
h1h4) around 200 Hz, 400 Hz, 600 Hz and 800 Hz (always ±20 Hz
due to the filter bandwidth of 50 ms for the FFT calculation, which caused
frequency steps of 20 Hz during analysis). Peak energies occurred within h3
and h1 (the fundamental frequency; Fig.
5A). The ABR waves evoked by this sound showed spectral peaks
corresponding to h1h4 of the stimulus and three further peaks at
frequencies that would correspond to h5h7.
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The sound pulses of B. modesta were low-pitched, rather broadband and showed no harmonic structure. The peak energy of the two-pulsed knocking sound was around 469 Hz (Fig. 5B). By contrast, the peak spectral energy of the corresponding ABR traces was around 156 Hz. The overall shapes of both spectra did not fit to each other and no clear correspondence in spectral peaks was detected (which is also partially because broadband spectra have no clear peaks). The last pulse of the conspecific sound consisting of three pulses was also rather broadband but with its peak energy at a lower frequency of about 273 Hz (Fig. 5C). In this case, ABR spectrum shape more closely paralleled the stimulus spectrum, especially in the frequency range of the stimulus with the main energy content between 230 Hz and 430 Hz; both ABR spectra, however, were relatively similar to each other.
The sound pulse of L. gibbosus was broadband with a peak energy at 547 Hz (Fig. 5D). This peak energy corresponded to a spectral peak of the ABR. The stimulus and ABR spectra shapes were quite parallel above 400 Hz, especially between 470 Hz and 840 Hz where the stimulus had its main energy content.
Representation of heterospecific sounds
Control tests using heterospecific sounds were performed in order to test
(1) whether the differences observed between L. gibbosus and the
hearing specialists were due to the sound itself or to differences in auditory
processing and (2) whether or not the fine temporal representation observed in
hearing specialists was restricted to conspecific sounds. Sounds of T.
vittata, L. gibbosus and B. modesta (three-pulsed sound) were
presented at an SPL of 100±1 dB to four specimens of P.
pictus. The catfish showed a similar temporal response pattern to
heterospecific sounds (Fig. 6).
The onsets of single sound pulses were highly correlated to the onsets of the
first negative peaks of the corresponding ABR waves (r=1,
P<0.001 for all the sounds tested). Similar to the responses to
conspecific sounds, brainwave amplitudes were not correlated significantly to
amplitudes of the sound pulses of B. modesta and of T.
vittata (r=0.423, P=0.164; r=0.248,
P=0.242, respectively). For the sound of L. gibbosus, a
negative correlation was observed (r=0.560,
P=0.024).
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In contrast to P. pictus, L. gibbosus showed irregular response patterns to the high-pitched stridulation sound of P. pictus and T. vittata (Fig. 7). Repeatable responses to these sounds could only be elicited at very high SPLs (120 dB for the sound of T. vittata and 129 dB for the stridulation sound of P. pictus) in all four individuals tested. Only one out of four individuals showed a response reflecting the temporal structure of both sounds. The responses to the sound of B. modesta (at an SPL of 113 dB) consisting of three pulses were more consistent among individuals. Similar to the responses to conspecific sounds, brainwaves were very long, lasting approximately 5090 ms. As the pulse period within the sound was much longer than in the sound of L. gibbosus, separate responses to each of the sound pulses were detectable, and the sound's temporal structure was reflected within the ABR (r=1, P<0.001). No correlation between amplitudes was performed because it was not possible to choose standardizable and identifiable measuring points in the diverse brainwaves due to the very long responses to the sound pulses. These results show that the difference between L. gibbosus and the hearing specialists is not due to the conspecific sound stimulus itself and that this species has difficulty in detecting high-pitched sound.
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Auditory sensitivity to conspecific sounds
Mean (±S.E.M.) hearing thresholds for conspecific sounds
were 64.1±1.7 dB re 1 µPa in P. costatus, 62.9±0.6
dB for stridulation sounds and 75.7±1.2 dB for drumming sounds in
P. pictus and 90.8±3.1 dB in T. vittata
(Fig. 8). In B.
modesta, the mean hearing threshold for the broadband sound consisting of
three short pulses was 77.3±1.3 dB and that for the sound consisting of
two longer pulses was 83.5±0 dB. In L. gibbosus, the mean
hearing threshold for the natural sounds was 97.9±1.2 dB, whereas for
the second test, lacking pulses 2 and 3, it was 95.7±1.9 dB.
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In all hearing specialists, hearing thresholds were 2656 dB under
the minimum SPL of conspecific sounds calculated for each species from
previous studies (Ladich,
1998,
1999
;
Wysocki and Ladich, 2001
) and
4165 dB below the averaged SPLs of the conspecific sounds emitted by
the fish (Fig. 8). No SPL
measurements were available for L. gibbosus sounds.
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Discussion |
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Auditory sensitivity to conspecific sounds
The auditory sensitivity to conspecific sounds of all hearing specialists
was comparable with previously measured tone burst thresholds of audiograms in
the most sensitive hearing range (Ladich
and Yan, 1998; Ladich,
1999
). Of all the species tested, P. pictus had the
highest sensitivity to stridulation sounds. This high sensitivity to broadband
pulses fits well to tone burst audiograms, which are quite flat in the high
frequency range (5003000 Hz; lowest hearing thresholds of about 67 dB
re 1 µPa in this range; Ladich,
1999
). The lower sensitivity to the low-pitched drumming sounds
(approximately 13 dB less sensitive compared with the stridulation sounds)
corresponds also to the lower sensitivity at lower frequencies (below 500 Hz;
lowest thresholds of approximately 74 dB) revealed by the audiogram
(Ladich, 1999
). B.
modesta also showed a different sensitivity to both sound stimuli tested,
which were broadband and relatively low-pitched but differed in their peak
frequencies (273 Hz versus 469 Hz) and duration of the single pulses.
The pulses to which the loaches were less sensitive by about 6 dB were
approximately twice as long as the pulses of the second sound tested. In ABR
audiometry, short stimuli with an abrupt onset are known to be more efficient
in evoking auditory potentials than stimuli with a long rising time
(Hall, 1992
). A single unit
study on the catfish Ictalurus nebulosus
(Plassmann, 1985
) revealed the
existence of two different main types of neurons in the medulla and
mesencephalon, a non-adapting (tonic) type and a fast-adapting type that
showed a steadily declining response with increasing stimulus rise time.
Beyond frequency effects, similar response characteristics of neuron
populations in the loaches might also be responsible for the different
detection thresholds observed (see Fig.
2). Trichopsis vittata showed less sensitivity than the
otophysines; this corresponds to the most sensitive frequency range in the
audiogram of this species.
A comparison was made between hearing thresholds and the SPLs of sounds emitted by the hearing specialists and calculated to a similar distance to the fish (3 cm) as the calibration of the stimulus underwater. This comparison showed that even the minimum sound levels measured were at least 25 dB and up to 60 dB above the hearing thresholds of these sounds. This points to a high detectability by the fish, especially because sound communication in these species most likely occurs at short ranges.
The sunfishes showed the least sensitivity to conspecific sounds. The SPL
hearing threshold of 97 dB, however, is about 20 dB more sensitive than that
in response to tone bursts in the range of the main sound energies
(300600 Hz) found in the closely related species Lepomis
macrochirus by other authors using a very similar ABR measuring technique
(Scholik and Yan, 2002).
Beyond species differences, this might be due to the very different type of
acoustic stimuli used. As hearing generalists are not pressure sensitive, and
can only detect the particle motion component of a sound, it might not seem
correct to measure their sensitivity in SPLs or to make comparisons. The main
goal of this study, however, was to investigate how conspecific sounds are
represented in the auditory system, in particular with regard to their
temporal structure in different species, and less so to determine absolute
sensitivity, which we therefore will not discuss further.
Representation of temporal patterns of conspecific sounds in the
brainstem of teleosts
Many natural sounds, particularly in a noisy environment such as water, are
broadband but with distinct temporal patterns. This might promote the
reliability of sound propagation in acoustic communication, where the spectral
content of the signal is often distorted because of absorption; this variously
enhances or cancels out particular frequencies depending on water depth and
the pressure release surface (Parvulescu,
1964). Therefore, a broad sound spectrum is thought to guarantee
the maximal acoustic signal under all water conditions
(Gerald, 1971
). In shallow
marine waters, the most reliably propagated sound characteristic of
damselfishes proved to be the pulse period
(Mann and Lobel, 1997
). Many
fish sounds are indeed broadband pulsed sounds, and temporal patterns seem to
be important factors for communication and species recognition in
damselfishes, sunfishes and cods (Spanier,
1979
; Gerald,
1971
; Hawkins and Rasmussen,
1978
).
A recent psychophysical hearing study in Pollimyrus
(Marvit and Crawford, 2000)
also concluded that the temporal discrimination abilities were sufficient not
only to discriminate between the calls of its own and closely related species
but most probably even between individuals. In Pollimyrus adspersus,
some neurons of the torus semicircularis in the midbrain were found to show
selective responses for a narrow range of ICIs, whereas another group of
neurons was classified as non-selective within the 1080 ms ICI range
tested. The distribution of best ICIs overlapped the 550 ms range of
communication sounds for this species
(Crawford, 1997b
). In all
species investigated during the present study, with the exception of the
hearing generalist L. gibbosus, each pulse within a sound elicited a
separate ABR wave. Thus, independent of sound length, the temporal pattern was
reflected at the level of the brainstem and is therefore a potential carrier
of information in hearing specialists. However, this ability to exactly
reflect temporal patterns is not restricted to conspecific sounds. In P.
pictus, for example, the auditory system also reflected the temporal
structure of heterospecific sound stimuli. In their natural environment,
fishes are confronted with diverse sounds besides intraspecific communication
sounds (e.g. from prey and predators) that also provide important information.
Therefore, hearing abilities should not be limited to conspecific sounds.
Preliminary tests showed that the auditory system of hearing specialists
responded similarly to temporal patterns of heterospecific sounds. In a
previous study on temporal processing of double clicks, vocal as well as
non-vocal fishes showed similar temporal resolution abilities
(Wysocki and Ladich, 2002
).
The high correlation between stimulus pulse onsets and onsets of corresponding
ABR waves indicates that the auditory system closely followed the temporal
structure of sounds. This implies that individual variations can be
recognized. In croaking gouramis, for example, the pulse periods within a
double pulse differ due to morphological asymmetries between fins, which was
also the case in our sound stimulus. Such asymmetries in absolute pulse
periods or even tendencies to produce triple pulses (L. E. Wysocki, personal
observation) make individuals quite differentiable.
Variations in latencies to the stimulus pulses were correlated to the pulse
amplitudes of conspecific sounds. Increasing ABR latency with decreasing SPL
is a common phenomenon in ABR audiometry of mammals (e.g.
Supin and Popov, 1995) and
fishes (e.g. Kenyon et al.,
1998
; Kratochvil and Ladich,
2000
). The present study shows that this phenomenon also occurs
within a series of sound pulses.
In the sunfish, a hearing generalist, the representation of temporal
information in the brainstem was less clear. ABRs to a stimulus pulse are very
long compared with specialists (several dozen ms). This might be due to
fundamental differences compared with specialists either in the auditory
periphery (lack of accessory hearing structures) or at more central levels of
the auditory system. Testing the responses to a modified sound of only the
first and the last pulse (pulse period approximately 88 ms) yielded two
separate responses. Subtracting the responses to the two-pulsed sound from
those to the four-pulsed sound revealed a remaining response to the mid-sound
pulses. This approach was successfully applied in dolphins and fishes to
determine whether an ABR waveform consisted of two responses to separate
clicks that are superimposed or of only one response
(Supin and Popov, 1995;
Wysocki and Ladich, 2002
);
waveform subtraction is also a validated procedure in human ABR audiometry
(Burkhard and Deegan, 1984). We can therefore assume that each of the four
pulses within the tested conspecific sound stimulus contributed to the evoked
response. This is supported by the findings of Gerald
(1971
), who showed in
behavioral playback studies that sunfishes are, to a certain extent, able to
selectively respond to conspecific sounds that mainly differed in their time
domain. In order to test whether this difference to hearing specialists was
due to differences in audition or simply to the type of sound tested, control
experiments were performed using heterospecific sounds as stimuli. The
responses of L. gibbosus to the low-pitched sound of B.
modesta were comparable with the responses to conspecific sounds.
Brainwaves lasted several dozens of ms but, because the pulse period of the
sounds was long enough, three separate responses to each of the sound pulses
were detectable and the temporal structure of the sounds was well represented
by the auditory system. These findings agree with those obtained using the
conspecific sound and its modification. By contrast, the individual sunfish
showed an irregular response pattern to the high-pitched sounds of P.
pictus and T. vittata. A repeatable response was obtained only
at very high SPLs, and the temporal structure of the sound was only reflected
clearly by one individual. We assume that only the low-frequency component of
the sounds elicited the ABRs because hearing generalists have a limited
hearing range, whereas stridulation sounds of P. pictus and croaking
sounds of T. vittata have their main energies above 1 kHz.
Representation of intensity and spectral content of sound
In contrast to the pulse periods, the correlation between pulse amplitudes
and amplitudes of the corresponding ABR wave was significant in only two out
of four species and close to significance in T. vittata. This can
mean that temporal structure, beyond sound intensity, might play a role in
assessing conspecifics. Ladich
(1998) showed in behavioral
tests that overall sound intensity is one factor influencing the outcome of
agonistic interactions in croaking gouramis. McKibben and Bass
(1998
) demonstrated in
playbacks with tonal stimuli that female plainfin midshipman (Porichthys
notatus) preferentially approached the more intense of two signals that
differed by just 3 dB.
Other sound characteristics are certainly also important in diverse
species. The dominant frequency of sounds (which is often correlated directly
to body mass, depending on the sound-producing mechanism) is known to play a
role during mate choice in damselfishes
(Myrberg et al., 1986) and for
the outcome of aggressive interactions in croaking gouramis
(Ladich, 1998
). In the present
study, spectral comparisons between ABR components and sound stimuli were only
performed for low-pitched sounds with main frequencies below 1 kHz because of
the filter settings of the electrode preamplifier. The clearest result was
obtained for the drumming sound of P. pictus, which showed a harmonic
structure. The fundamental frequency of the sound was predominantly present in
the ABR spectrum. In addition to the spectral peaks at h1h4 of the
stimulus, response components were consistently observed in all individuals at
frequencies that presumably corresponded to h5h7. A similar phenomenon
has been observed in human ABR spectra to two-tone approximations of
steady-state vowels (Krishnan,
1999
). In humans, spectral peaks of the responses were not only
observed at the formant frequencies of the sounds tested but also at
frequencies that corresponded to harmonics of one formant of the stimulus and
were not present in the stimulus spectrum. Spectral peaks within ABRs matching
to spectral peaks of sound stimuli reflect temporally locked activities of
populations of neurons to the frequency components of the stimuli. Neural
phase-locking plays an important role in encoding spectral features of sounds
(Krishnan, 2002
).
In L. gibbosus, most similarities between stimulus and ABR spectra were found in the range of the main sound frequencies. As the sound is rather broadband, it is difficult to correlate particular spectral peaks to each other. Nonetheless, the overall spectral similarity in this particular frequency range indicates some influence of frequency components on the auditory system of the fish.
In B. modesta, it was not possible to interpret ABR spectral peaks
as specifically representing the frequency components of the sound stimuli.
However, the ABR waveforms differed in response to both sound stimuli (see
Fig. 2B). This may be induced
by a different activation pattern in the neurons and indicates that the
perception of differences probably also relies on other characteristics such
as pulse duration or envelope shape of the stimulus. There is evidence that in
plainfin midshipman the different frequency of acoustic beats and the
modulation frequency of amplitude-modulated signals are coded differently by
neurons in the auditory midbrain, even if it is the same frequency
(Bodnar and Bass, 1997). This
could permit discrimination of beats (due to concurrent vocalizations of males
during the breeding season) from other amplitude-modulated like signals.
Note that ABR waves only reflect the first steps of signal processing in
the brainstem up to the midbrain (Corwin,
1981) and that various response parameters change along the
central auditory pathway up to `higher' brain levels (e.g. an increase in
auditory sensitivity and in transient responses to stimulus onset and offset;
Feng and Schellart, 1999
). We
therefore conclude that, besides temporal patterns, frequency and intensity
characteristics can also be transmitted by acoustic signals. Together, these
provide complex information for the fish during acoustic communication.
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References |
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