J Comp Physiol A (1998) 183: 265±271
Ó Springer-Verlag 1998
ORIGINAL PAPER
E. D. Lindquist á T. E. Hetherington á S. F. Volman
Biomechanical and neurophysiological studies on audition in eared and earless harlequin frogs (Atelopus)
Accepted: 4 April 1998
Abstract Tissue displacement of various body surfaces and the auditory midbrain sensitivities to sound were measured in Atelopus species with or without a tympanic middle ear (``eared'' and ``earless'', respectively). Tissue displacement (vibration) of body regions was measured by laser Doppler vibrometer . The body wall directly overlying the lung is most dramatically displaced by sound pressure in all species tested. The otic (lateral head) region showed low displacement in earless species, but signi®cant displacement to high-frequency sound in eared species. Peak tissue displacement of the body wall occurred within the frequency range of each species' advertisement vocalization. Peak tissue displacement of the otic region of the eared species also occurred within these frequencies. Multi-unit neurophysiological recordings of the auditory midbrain (torus semicircularis) also were obtained. Auditory sensitivity curves showed three distinct regions of sensitivity at low, middle, and high frequencies, the latter located within the frequency range of each species' advertisement vocalization. The correlation between auditory midbrain sensitivity and tissue displacement of the body wall region at advertisement vocalization frequencies, suggests that the body wall/lungs serve as the route of sound transfer to the inner ear in earless species and possibly in the eared species as well. Keywords Anura á Atelopus á Torus semicircularis á Tissue displacement á Auditory pathways
E.D. Lindquist1 (&) á T.E. Hetherington á S.F. Volman Department of Zoology, The Ohio State University, 1735 Neil Ave., Columbus, OH 43210, USA e-mail:
[email protected]/
[email protected], Tel.: +1-614-292-0832; Fax: +1-614-292-2030 Present address: Department of Natural Sciences and Mathematics Lee University, North Ocoee St., Cleveland, TN 37320-3450 USA e-mail:
[email protected] Tel.:+1-423-614-8282; Fax: +1-423-614-8295
1
Abbreviations LDV laser Doppler vibrometer á SPL sound pressure level á TS torus semicircularis
Introduction Vocalizations play an important role in the mating and territorial behavior of most anuran amphibians (Wells 1977), and most species have well-developed tympanic middle ears, consisting of an external tympanum, air®lled middle ear cavity, Eustachian tubes, and auditory ossicle, for sound reception (Wever 1985; Jaslow et al. 1988). However, several species of anurans entirely lack a tympanic middle ear, an anatomical condition often referred to as ``earless,'' although they have standard inner ears containing well-developed auditory sensory organs (Wever 1985; Jaslow et al. 1988). Despite the lack of a tympanic middle ear, many earless species of frogs produce a variety of vocalizations, suggesting that they are utilizing alternative pathways of sound reception for detecting their calls (Jaslow et al. 1988; Cocroft et al. 1990). Most species of harlequin frogs of the genus Atelopus entirely lack tympanic middle ears ( earless), but some species possess slightly reduced tympanic middle ears ( eared; McDiarmid 1971). The latter middle ear con®guration includes all the standard anatomical components except a specialized tympanum. In the eared species of Atelopus, the extracolumella attaches to unspecialized skin bordering the lateral edge of the middle ear cavity (E. D. Lindquist et al., personal observation). Both eared and earless species of Atelopus produce a variety of calls (Cocroft et al. 1990), and Lindquist and Hetherington (1996) demonstrated that the earless A. zeteki displays behavioral responses to conspeci®c vocalizations in the ®eld. Wever (1985) was able to measure cochlear microphonic responses to airborne sound in several species of earless Atelopus, and studies of midbrain responses by Jaslow and Lombard (1996) found that the earless A. chiriquiensis is surprisingly sensitive to sound. The level of auditory sensitivity
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observed suggests that eective nontympanic pathways of sound transfer are present in these species. There is evidence that some frogs may use the lateral body wall and lungs as a nontympanic route for sound reception (Narins et al. 1988; Ehret et al. 1990, 1994, Hetherington 1992). Measurements of the tissue displacement of body surfaces using a laser Doppler vibrometer have demonstrated that the lateral body wall directly over the lungs is easily displaced by sound (Narins et al. 1988; Jùrgensen 1991; Hetherington 1992; Ehret et al. 1994). In species that possess complete tympanic middle ears, pressure waves within the lungs can pass forward through the glottis, mouth cavity, and Eustachian tubes to the middle ear cavity and aect tympanic motion (Ehret et al. 1990; Jùrgensen et al. 1991). Ehret et al. (1994) demonstrated that attenuation of high-frequency sound along the lung-eardrum pathway is pronounced, so that the contribution of the lung to tympanic displacement is restricted to relatively low frequencies. It has been hypothesized that the body wall and lungs can directly transfer sound energy to the inner ear of earless species of frogs (Narins et al. 1988; Hetherington 1992), although there has been no experimental demonstration of the precise route. The presence of both eared and earless species within the genus Atelopus provides a unique opportunity to study dierent strategies of sound reception (tympanic versus nontympanic) in closely related and anatomically similar animals. This study was to analyze both the auditory midbrain sensitivity and the tissue displacement of various anatomical surfaces in eared and earless species of Atelopus. Potential peripheral sound pathways were analyzed by directly correlating the acoustic properties of the dierent body surfaces with auditory midbrain sensitivities.
Materials and methods Animals Laser vibrometric and neurophysiological tests were conducted on A. ¯avescens, an ``eared'' species, and on two undescribed ``earless'' species, Atelopus sp. (Chingaza) from Parque Natural Nacional Chingaza, Colombia and Atelopus sp. (Nusagandi), from Parque Nacional Nusagandi, Kuna Yala, Panama. Measurement ranges for each species/sex are as follows: A. ¯avescens # 28 mm/1.3 g and $ 29±33 mm/1.8)1.9 g, Atelopus sp. (Chingaza) # 27± 33 mm/1.4±1.8 g and $ 32±39 mm/1.5±1.3.1 g, Atelopus sp. (Nusagandi) # 27±28 mm/1.1±1.5 g and $ 31 mm/1.7 g. Histological examination has demonstrated that A. ¯avescens lacks a specialized tympanum but possesses a well-developed middle ear cavity, Eustachian tubes, and auditory ossicle while the earless Atelopus species completely lack these structures (McDiarmid 1971; E.D. Lindquist et al., personal observation). All frogs were wildcaught adults and were obtained legally from the country of origin. Chingaza frogs are a high-elevation species (4500 m above sea level) and were kept in a standard 40-gallon terrarium in a refrigerated room (7±15 °C). Atelopus sp. (Nusagandi) and A. ¯avescens are low-elevation species (500±1700 m above sea level) and were kept in standard 10-gallon and custom-made 150-gallon terraria at room temperature (23 °C) at the Ohio State University, Columbus, Ohio, USA. The animals were maintained on a 12:12 light/dark cycle.
Laser vibrometry Laser Doppler vibrometric (LDV) experiments were performed on animals lightly anesthetized by immersion in a 1% tricaine methanesulfonate solution (MS-222) so that they sat motionless during experimentation. Animals were then placed in a sound-attenuating box where a neon-helium laser beam from a LDV (Polytec OFV 1000) was focused on a small re¯ector (0.3 mm ´ 0.3 mm) placed on certain body surfaces. LDV measurements were made at three body surface points: the lateral body wall directly over the lung, the otic region over the inner ear region, and rostrum (snout midway between the external nares and eyes). The latter represented a control body surface. Animals were frequently moistened to prevent the skin surfaces from drying. Adult frogs [A. ¯avescens {1#:4$}; Atelopus sp. (chingaza) {2#:2$}; Atelopus sp. (Nusagandi) {3#:1$}] were used in all LDV experiments. Computer-generated sinusoidal pure tones were ampli®ed and delivered to frogs through a full-range 25.4-cm speaker using Wave SE acoustic software. Tones separated by roughly one-third-octave steps (0.16/0.2/0.25/0.315/0.4/0.5/0.63/0.8/1.0/1.2/1.6/2.0/2.5/3.15/ 4.0/5.0/6.3 kHz) were broadcast continuously at a distance of 30 cm from the test subject. The frogs were within the speaker's near ®eld at frequencies below 500 Hz. LDV measurements were made on body surfaces ipsilateral to the speaker. Sound pressure level (SPL) was maintained at 90 dB (RMS-slow) and monitored with a 1.27-cm condenser microphone (BruÈel & Kjaer Type 4155) positioned 2 cm above the dorsum of each animal and connected to a sound-level meter (BruÈel & Kjaer Type 2230) and third octave/ octave ®lter set (BruÈel & Kjaer Type 1625). LDV output voltage for each tested frequency was measured on a wave analyzer (Hewlett Packard 3581A), averaged across individuals of each species, and converted to velocity. Velocities were then converted to average displacement values (dividing velocity by 2pf, where f frequency). Individual animals were tested once and values were obtained for each anatomical region at every frequency. The tissue displacement (vibration amplitude) of each anatomical region for each individual was averaged at each frequency to yield an average tissue-response curve for each species.
Neurophysiology Surgeries for neurophysiological experiments were performed on animals anesthetized by immersion in 1% MS-222. The dorsal surface of the midbrain was exposed through an opening in the parietal bones of the skull. After surgery, animals were immobilized for multi-unit midbrain recordings with an intramuscular injection of of 15 ll g)1 body weight of 9 mg ml)1 d-tubocurarine chloride. The required dosage for Atelopus is unusually high for anurans. Individual frogs were placed on an experimental stage located in a sound- and vibration-attenuating booth. Animals used in the neurophysiological experiments had to be completely immobilized and were positioned in a resting posture less upright that the LDV experiments. A tungsten electrode (FHC, 11±13 MW) was inserted 220±775 lm deep into the torus semicircularis (TS, auditory midbrain) by use of a stepping-motor microdriver (M. Walsh Engineering, UD-220). Computer generated sinusoidal tone pips were ampli®ed and delivered to frogs through a full-range speaker using custom acoustic software (as described in Volman 1996). Auditory stimuli lasted 250 ms and had 5-ms rise and fall times and were repeated every 3.5 s. Multicellular responses were ampli®ed with an AC ampli®er (A-M Systems 1800) and played on a speaker located outside the experimental booth. Hearing thresholds for each species were determined by auditory monitoring of multiunit responses as the amplitude of tone pips was manually attenuated by 1-dB increments. TS threshold sensitivities were obtained at frequencies ranging from 100 Hz to 5000 Hz at 100-Hz intervals. Presentation order of stimulus frequencies was randomized. Atelopus species are small and have been found to be relatively fragile during neurophysiological experiments compared to other anurans, and hence, recordings, from the TS were conducted rather than using the eighth nerve. The midbrain is quite small, and in
267 some individuals only one complete electrode pass was possible before TS responses decreased. Midbrain responses were measured from two female A. ¯avescens (two complete curves from one animal and one complete and one partial curve from another individual), six male and one female Atelopus sp. (Nusagandi) (three complete curves from one individual, two curves from each of two other individuals, and a single curve from each of the remaining four individuals), and ®ve male Atelopus sp. (Chingaza) (three complete curves from one individual, two curves from each of two other individuals, and a single curve each from another two individuals). The minimum audible thresholds from each individual were averaged to yield a mean audibility curve for each species. A minimum threshold curve for each species also was plotted by incorporating minimum audible thresholds among all individuals of that species at all sound frequencies.
Results Laser vibrometry The frequency response of tissues varied dramatically among the anatomical regions tested. Tissue displacement of the body wall directly over the lung was the highest tested anatomical region for all species (Fig. 1A± C). The body wall showed high displacement at low frequencies below about 400 Hz and even greater displacement at higher frequencies centered around 2500 Hz (Fig. 1A±C). The peak tissue displacement for the high-frequency range falls within the frequency range for the species' advertisement vocalization for each species (Fig. 1A±C). The rostral (control) region of the head provided consistently low vibration amplitudes at all test frequencies for each species (Fig. 1A±C). The mean tissue displacement across most test frequencies of the body wall was generally greater than that of the rostrum. The tissue displacement of the otic region was low for the two earless Atelopus sp. over the entire range of frequencies (Fig. 1B±C). Displacement of the otic region of the eared A. ¯avescens was similarly low at most frequencies, but markedly greater around 2500 Hz and thereby fell within the frequency range of the species advertisement vocalization (Fig. 1A). Neurophysiology TS threshold curves showed three distinct frequency regions of sensitivity in both the eared and earless species of Atelopus: a low-frequency (100±400 Hz) region, a middle-frequency (around 1500 Hz) region, and a highfrequency (around 2000±2500 Hz) region (Fig. 2A±C). In all cases, the high-frequency range of auditory sensitivity falls within the frequency range of the species' advertisement vocalization for each species (Fig. 2A±C). Figure 3 shows a comparison of the average auditory threshold curves for eared and earless species of Atelopus. The auditory sensitivity measured for the two specimens of A. ¯avescens demonstrate that this eared species is slightly (8±13 dB SPL) more sensitive to high-
Fig. 1A±C Mean tissue displacement of anatomical surfaces: black line, body wall region (lung) with standard error (of x) bars; shortdashed line, otic region (with standard error bars in A. ¯avescens); long-dashed line, rostral region. A A. ¯avescens (n 5). B Atelopus sp. (Chingaza) (n 4). C Atelopus sp. (Nusagandi) (n 4). Horizontal bar represents the frequency range of the species' advertisement vocalization (Lescure 1981; C. Navas, personal communication; R. IbaÂnÄez, personal communication; respectively)
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pear to have greater sensitivity to low-frequency (below about 400 Hz) sound than does A. ¯avescens.
Discussion Comparative TS sensitivities Overall, the shape of TS sensitivity curves in eared and earless Atelopus was similar, although dierences in absolute sensitivity were apparent at certain sound frequencies. The eared A. ¯avescens was roughly 8±13 dB more sensitive at high frequencies (around 2000±2500 Hz) compared to the earless Nusagandi and Chingaza Atelopus sp. (see Fig. 3). However, earless species showed higher auditory midbrain sensitivity (by as much as 24 dB) at low frequencies below 500 Hz (see Fig. 3). The general shape of the auditory midbrain sensitivity curves was similar for both eared and earless species. All specimens showed enhanced sensitivity to low-frequency sound below about 400 Hz, to middle-frequency sounds around 1500 Hz, and to higher-frequency sounds around 2000±2500 Hz. These three most sensitive regions of the threshold curves likely represent responses from dierent auditory hair cell populations. The low-, middle-, and high-frequency sensitivities likely are associated with the saccule and/or amphibian papilla, the amphibian papilla, and basilar papilla, respectively. This pattern is re¯ected in many other anuran species (Feng et al. 1975; Moat and Capranica 1976; Wilczynski and Capranica 1984; Zakon and Wilczynski 1988). The highfrequency TS sensitivities of both the eared and earless Atelopus are tuned to the range of frequencies of the species' advertisement vocalization (Fig. 2A±C).
Fig. 2A±C Multiunit auditory sensitivity-threshold curves recorded from torus semicircularis: black line, mean auditory threshold with standard error (of x) bars; dashed line, minimum auditory threshold for species. A A. ¯avescens (n 2). B Atelopus sp. (Chingaza) (n 5). C Atelopus sp. (Nusagandi) (n 7). Horizontal bar represents the frequency range of the species' advertisement vocalization (Lescure 1981; C. Navas, personal communication; R. IbaÂnÄez, personal communication; respectively)
frequency sound (around 2000±2500 Hz) than both earless species. Earless Atelopus on the other hand ap-
Fig. 3 Mean torus semicircularis (TS) auditory sensitivity for eared and earless species compared. F (thick solid line) A. ¯avescens; C (thin solid line) Atelopus sp. (Chingaza); and N (dashed line) Atelopus sp. (Nusagandi)
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Correlations between LDV measurements and TS sensitivity This study demonstrated that the lateral body wall overlying the lungs is easily displaced by sound in both eared and earless species of Atelopus. The body wall showed a low-frequency range of tissue displacement below about 400 Hz and a higher-frequency range of tissue displacement around 2000±2500 Hz. The latter zone of tissue displacement corresponds well to the range of frequencies in the species' advertisement vocalization (Fig. 1A±C). In LDV experiments, the frogs were positioned within the speaker's near ®eld at frequencies below 500 Hz. Therefore, the tissue displacement of the body wall in this frequency range may have been accentuated by additional displacement produced by near-®eld eects. Indeed, this study found that the body wall of Atelopus showed much higher amplitude motion at such low frequencies than frogs of the genera Hyla, Rana, and Eleutherodactylus as reported by Jùrgensen (1991) and Jùrgensen et al. (1991). The latter species showed comparable high-frequency peaks in body-wall motion, but lacked the high-amplitude displacements at low frequencies. Although it is possible that the low-frequency motion of the body wall of Atelopus is related to near-®eld eects, the amplitude of body-wall motion at these low frequencies was greater than that of the head and ear region, suggesting that the body wall over the lung indeed is more responsive to these frequencies than other body surfaces. Generally, the body wall showed greater displacement amplitude to sound than the rostral region of all species and the otic (lateral head) region of the earless species for most frequencies. The body wall area of the eared A. ¯avescens also showed greater displacement amplitude than the otic region of this species across the full range of frequencies, although the otic area did show a high-frequency peak in tissue displacement at about 2500 Hz. The latter peak in otic displacement was nontheless signi®cantly lower than the same high-frequency peak in body wall displacement. Therefore, both the tympanum (otic region) and body wall of the eared A. ¯avescens displayed peaks in displacement amplitude at frequencies around 2000±2500 Hz, although the body wall showed higher amplitude responses. Figure 4A±C shows the good correspondence between body-wall displacement (and otic displacement for A. ¯avescens) and the high-frequency region of sensitivity of the TS in Atelopus. The structural basis of hearing in eared and earless species. The surprising similarity in auditory midbrain sensitivities between eared and earless species of Atelopus suggests that nontympanic pathways of sound reception can be quite eective compared to standard tympanic middle ear. The presence of a tympanic middle ear typically
Fig. 4A±C Correlation between mean TS auditory threshold and tissue displacement amplitude of the body wall/lung region: upper thin solid line, mean TS auditory threshold; heavy solid line, mean displacement of body wall/lung surface; lower thin solid line (only in A. ¯avescens), mean displacement of otic region surface. A A. ¯avescens, B Atelopus sp. (Chingaza). C Atelopus sp. (Nusagandi). Horizontal bar represents the frequency range of the species' advertisement vocalization (Lescure 1981; C. Navas, personal communication; R. IbaÂnÄez, personal communication; respectively)
would be expected to impart better sensitivity to highfrequency sound, and this was indeed observed, al-
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though the increase in sensitivity (8±13 dB) was not dramatic. A. ¯avescens lacks a specialized tympanum, however, and comparison of the high-frequency auditory sensitivity of the earless species to that of a species with fully developed tympanum may provide more pronounced dierences. The body wall of all of the species examined showed patterns of tissue displacement that correlated with the low-frequency and high-frequency areas of peak TS sensitivity. The middle-frequency peak in TS sensitivity was not re¯ected by pronounced displacement amplitude of the body wall. Most studies of body wall/lung displacement to sound have emphasized how lung-borne sound can be transferred through the glottis and Eustachian tubes and directly modify tympanic motion (Narins et al. 1988, Ehret et al. 1990, 1994). Given that earless species of Atelopus entirely lack a tympanic middle ear, including Eustachian tubes, sound energy penetrating the lateral body wall and entering the lung must be directly transferred to the inner ear. Narins et al. (1988) proposed that one potential route could involve the transfer of sound energy from the lung to ¯uid spaces surrounding the spinal column, and from there to the endolymphatic sac and into the inner ear. Several possible pathways exist, however, and additional research is needed to isolate the routes of sound transfer. Ehret et al. (1994) found that high-frequency signals passing from the lungs to the middle ear cavity were greatly attenuated, and that this route was more eective for transmission of low-frequency signals. Our study demonstrates that earless species are on average 8±13 dB less sensitive to high frequencies than a species with a slightly reduced tympanic middle ear, and anurans with a specialized tympanum may be even more sensitive to high frequencies. Although the lung-inner ear pathway may be less eective at high frequencies than a tympanic middle ear, this study suggests that high-frequency signals nonetheless can be transferred from the lungs to the inner ear. The alternative would be that high-frequency sound might directly penetrate the lateral head tissues and pass to the inner ear, but the very low tissue displacement of head tissues to high frequencies observed in this study suggests that this is not the case. It is unclear how much of the auditory sensitivity of the eared A. ¯avescens depends on lung sound reception. As mentioned above, it has been demonstrated that sound acting on the body wall and lungs of frogs with tympanic middle ears can pass through the glottis and Eustachian tubes and directly produce tympanic motion (Narins et al. 1988; Ehret et al. 1990, 1994). However, nontympanic routes of sound transfer from the lungs to the inner ear that operate in earless frogs may function in eared species as well. Wilczynski et al. (1987) demonstrated that the Rana pipiens auditory nerve responses to sounds below about 500 Hz could be produced more eectively by sound transfer along nontympanic pathways. However, aspects of nontympanic sound reception may be body-size dependent, as the body wall and lungs of small anurans may vibrate more readily at high sound
frequencies (Hetherington 1992). As the species of Atelopus used in this study are smaller than R. pipiens, the eectiveness of their nontympanic sound transmission may extend to higher frequencies. Therefore, it is possible that body wall reception of sound can contribute to auditory responses over a broad range of frequencies in the eared A. ¯avescens. The greater auditory sensitivity of A. ¯avescens to high-frequency sounds compared to its earless congeners may result from two potential mechanisms. First, such sensitivity may be due to direct reception of these signals by the otic area and extracolumella/columella. Although the body wall actually vibrates more readily than the otic region to these high-frequency sounds, there may be pronounced attenuation of sound energy, especially at high frequencies, during transfer from the lungs. Second, sound transferred from the body wall and lung via the glottis and Eustachian tubes may increase otic/columellar responses to sound at these frequencies. Because the otic region of this species showed little tissue displacement at low-frequency sound, direct transfer of sound energy from the lungs to the inner ear seems the most likely explanation. The poorer auditory sensitivity of the eared A. ¯avescens to low frequency compared to the earless species is dicult to explain. Possibly the earless species possess specialization of either their nontympanic pathways or inner ear that enhance sensitivity to lower frequencies. The functional basis of middle ear loss in frogs The adaptive signi®cance of the dierences in middle ear morphology in the neotropical genus Atelopus remains speculative. Both eared and earless species commonly are found along streams, but the eared species tend to live in lowland areas and the earless species inhabit both lowland and montane regions. It has been proposed that the montane stream habitats are noisier, thereby making acoustic communication more dicult (Heyer et al. 1990) and minimizing the selective advantage of a tympanic middle ear. However, results from this and other neurophysiological studies demonstrate that earless Atelopus are sensitive to SPLs as low as 43±62 dB SPL, and ®eld studies have established that they respond behaviorally to conspeci®c vocalizations and can locate sound sources (Lindquist and Hetherington 1996). A better understanding of the functional and evolutionary basis of middle ear loss in anurans will best be achieved by a combination of behavioral and functional studies on closely related eared and earless species. Acknowledgements We wish to thank Carlos Navas and Roberto lbaÂnÄez DõÂ az for information on vocalizations of Atelopus sp. (Chingaza) and (Nusagandi), respectively. We also are grateful for the technical assistance of Kris Schuett and Jim Fox. We thank W. Wilczynski and two anonymous reviewers for their editorial assistance on an earlier version of this paper. These experiments comply with the ``Principles of animal care,'' publication NO.86-23, revised 1985 of the National Institutes of Health and also with the current laws of the state of Ohio, USA.
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