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Hair cell morphology and innervation in the basilar papilla of the emu (Dromaius novaehollandiae)
Hearing Research 121 (1998) 112^124
Hair cell morphology and innervation in the basilar papilla of the emu (Dromaius novaehollandiae) Franz Peter Fischer * Institut fuër Zoologie, Technische Universitaët Muënchen, Lichtenbergstrasse 4, D-85747 Garching, Germany Received 9 December 1997; revised 13 April 1998; accepted 18 April 1998
Abstract The emu, being a member of the rather primitive bird group of the palaeognathid Ratitae, may reveal primitives features of the avian basilar papilla. There are, however, no qualitative differences with the papillae of other birds such as the chicken or the starling. There are only quantitative differences in the continuous morphological gradients (such as hair cell height, stereovillar height) from neural to abneural, and from the base to the apex of the papilla. Only few (about two in the emu) afferent terminals and on average one efferent fiber contact each hair cell. Along the abneural edge, there is a population of hair cells that lack afferent innervation (short hair cells), suggesting that their function must lie in the papilla itself. There is thus a general pattern in the structures of the avian basilar papilla. In detail, however, a number of primitive characters were observed in the emu, as compared to advanced birds such as the starling and the barn owl. The hair cells are very densely packed and comparatively tall (up to 40 Wm in the apex). This anatomy correlates well with the good lower-frequency hearing (see Koëppl and Manley, J. Acoust. Soc. Am. 101 (1997) 1574^1584). The afferent nerve fibers contacting the hair cells within the basilar papilla are rather thick, and there are a large number of afferent fibers that contact more than one hair cell. The zone of hair cells without afferent innervation (short hair cells) along the abneural edge of the basilar papilla is rather narrow in the emu. z 1998 Elsevier Science B.V. All rights reserved. Key words: Bird; Emu; Cochlea; Hair cell; Innervation; Evolution
1. Introduction In the last decade, di¡erent research groups have shown that hair cells (HC) in the avian inner ear can regenerate, e.g. after acoustic trauma, in contrast to the situation in the mammalian cochlea (e.g. Cotanche, 1987 ; Corwin and Cotanche, 1988; Ryals and Rubel, 1988). In this new research area, the chicken has become the standard bird. The morphology, innervation and synaptic physiology of the HC in the chicken basilar papilla (BP) have been studied intensively (Cole and Gummer, 1990 ; Fischer, 1992; Ofsie and Cotanche, 1996 ; Zidanic and Fuchs, 1996; Fuchs, 1992; Fuchs and Murrow, 1992; Code and Carr, 1994; Kaiser and Manley, 1994). The ears of other avian species have been much less well investigated. * Tel.: +49 (89) 2891-3662 (3661); Fax: +49 (89) 2891-3674; E-mail: [email protected]
The inner ears of di¡erent bird species have several morphological and functional similarities, but also marked di¡erences from the mammalian organ of Corti. The avian hearing epithelium, the basilar papilla, is an elongated £at band. In contrast to the mammalian organ of Corti, there are no distinct hair cell rows like inner and outer hair cells (IHC and OHC). In birds, the hair cells occupy the whole surface, and are separated from each other by narrow supporting cells. Like mammals, birds have di¡erent forms of HC across the papilla, the extremes being the tall hair cells (THC) on the neural and the short hair cells (SHC) on the abneural side, with, however, a continuous transition in between. The mechanism of hearing is far less well understood in birds than in mammals (Klinke and Smolders, 1993; Manley et al., 1989; Manley, 1995). The comparison of anatomy and innervation pattern in the hearing organ of di¡erently specialized avian species is one approach to obtain a better insight into the relationship between
0378-5955 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 9 8 ) 0 0 0 7 2 - 0
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structure and function, between the anatomy of the hearing epithelium and its innervation on the one hand, and physiological data on the other hand. We studied, at the transmission electron microscopy (TEM) level, the patterns and gradients in HC morphology and innervation as compared to their position on the BP and thus to their characteristic frequencies. This provides the opportunity of obtaining a large and detailed body of systematic quantitative information. So far, we have studied the inner ear of the chicken (Fischer, 1992), the starling (Fischer et al., 1992) and the barn owl (Fischer, 1994a) in such detail. Frequency maps of avian BP are currently available for the chicken (Manley et al., 1987; Chen et al., 1994; Jones and Jones, 1995), the starling (Gleich, 1989), the pigeon (Klinke and Smolders, 1993; Smolders et al., 1995), the barn owl (Koëppl et al., 1993) and recently also for the emu (Koëppl and Manley, 1997). The Australian emu (Dromaius novaehollandiae) is considered to be a rather primitive bird (Feduccia, 1980 ; Carrol, 1988); it is a member of the palaeognathid line that separated early in the avian radiation from the neognathid birds. Its body structures show several primitive features (Cracraft, 1981). The study of its inner ear may possibly reveal primitive features in the morphology and innervation pattern of the avian hearing organ. Therefore we quantitatively analyzed the BP of the emu in a similar way as before in the chicken, the starling and the barn owl. 2. Materials and methods For this quantitative systematic study, ¢ve positions along the basilar papilla (BP) of a 4^5 day old (posthatch) emu (Dromaius novaehollandiae LATHAM 1790) were analyzed using serial TEM sections. Two papillae of additional individuals (a hatchling and a 70 week old emu) served as TEM controls to exclude the possibility that the analyzed specimen was abnormal. The hatchling was analyzed at ¢ve similar positions, the other one only at three positions. As the data were similar, only one BP (that of the 4^5 day old emu) is described here in detail. Some aspects of the BP of the emu hatchling were already included in a separate comparative study (Fischer, 1994b). Additional ears were studied at the light microscopic level. The emu was anesthetized with a combination of Chlorthesin and Nembutal, and electrical recordings were made from the acoustic nerve for several hours (Manley et al., 1997). Then the round window was opened and the columella removed. A small volume (a few ml) of cold ¢xative (5% glutaraldehyde in phosphate bu¡er, pH 7.4) was slowly introduced through the oval window and drawn out of the ear via the round window. The animal was then killed by an over-
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dose of anesthetic, and the ear was dissected free and placed overnight in chilled ¢xative. Then it was washed in chilled bu¡er, and post¢xed in 2% osmium tetroxide in bu¡er for 2 h. After dehydration in ethanol it was embedded in Araldit. The ¢xation protocol for the other ears was slightly di¡erent, but the ultrastructure and dimensions of BP and HC were very similar. Serial semithin and ultrathin sections were cut with a Reichert ultramicrotome. The semithin sections were stained with 1% toluidine blue in 1% borax solution, the ultrathin sections with uranyl acetate and lead citrate. The ultrathin series were at the positions 9%, 26%, 41%, 68% and 89% as measured from the base of the BP. This corresponds to frequencies of 3.3, 1.14, 0.7, 0.2 and 0.08 kHz, respectively (Koëppl and Manley, 1997). The sections were studied in a Jeol JEM 100 CX TEM at a primary magni¢cation of 2600U and every fourth section was photographed. In every session, an exact calibration of the TEM was carried out. The photographs were subsequently enlarged to 5000U. Details were studied at higher magni¢cation. All measurements given in this paper are for the ¢xed and embedded specimen, without correction for shrinkage (as previously in the other species). The HC and their nerve endings were drawn on transparent sheets. These were placed on top of each other, and, with the HC surface and the nucleus as landmarks, the sheets were used for the reconstructions. As the HC in the apex of the BP are slightly obliquely orientated and also often twisted, exact measurements of HC length were only possible from complete reconstructions from serial sections of known thickness. The numbers of nerve terminals were directly derived from these reconstructions. Synaptic areas were calculated from the length of contact zones and the thickness of the sections. Altogether, 107 HC and their innervation pattern were reconstructed (14 HC at the 9% position, 16 at the 27% position, 24 at the 41% position, 23 at the 68% position, and 30 at the 89% position). Ninety-nine HC could be used for the analysis of the innervation pattern. All procedures were in conformity with the Australian regulations for animal protection and were approved by the Animal Experimentation Ethics Committee of the University of Western Australia (64/93/93 and 180/94/94). 2.1. De¢nitions To avoid confusion, in this paper the three terms length, width and height are used as follows : `length' is measured parallel to the BP's length, `width' is measured parallel to the BP's width, and `height' is measured roughly perpendicular to both (e.g., along the HC's long axis).
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Fig. 1. Dimensions of the BP (a) and number of HC across the BP (b). Data points and smoothened lines. The abscissa represents the neural border of the BP.
3. Results 3.1. Dimensions of the emu basilar papilla Compared with most other birds (a marked exception being the barn owl; Smith et al., 1985; Fischer et al., 1988 ; Gleich et al., 1994; Manley et al., 1996), the dimensions of the emu's BP were in the upper range. The length of the BP was 4.65 mm (¢xed and embedded). The width ranged from about 80 Wm in the base to 463 Wm near the apical end (Fig. 1a). The number of HC across the BP was 13 in the base and 49 in the broadest part (near the apex). Taking the number of HC across the BP, the HC diameters, and the BP dimensions at the ¢ve positions into account, an overall number of about 16 500 hair cells was calculated for the 4^5 day old emu and 16 900 for the hatchling. A substantial proportion of the HC were placed over the neural limbus (Fig. 1b). The zone of the hyaline cells along the abneural border was remarkably broad and occupied up to 100 Wm of the width near the apex (Fig. 1a). 3.2. HC morphology along and across the BP Basal and apical HC had quite a di¡erent shape. Apical HC were tall, basal HC were much shorter. Apical HC had much active cytoplasm (cytoplasm without the cuticular plate), whereas basal HC generally had less active cytoplasm, especially those on the abneural side of the papilla. Over the width of the BP, taller HC on the neural side graded into shorter HC on the abneural side. Compared to other avian species, the HC in the emu were remarkably tall. Their width, however, was average. They were unusually densely packed, with only little space for the separating supporting cells, especially in the apical half of the BP, where the HC virtually touched each other. The ultrastructure of the HC and their synapses was similar to that in other avian species (e.g. chicken : Tanaka and Smith, 1978; Fischer, 1992; pigeon : Takasa-
ka and Smith, 1971 ; starling : Fischer et al., 1992; duck: Chandler, 1984) and will therefore not be repeated here in detail. Kinocilia were occasionally observed throughout the whole BP. Where they were vestigial, a basal body remained. As in the other avian species examined, contacts between neighboring HC (Fischer et al., 1991) were frequent in the apical half of the BP (see also Fig. 7). All four previously described contact types were found, including true membrane fusions. Altogether, of 34 contacts evaluated, 53% were of the touch type, 23% of the fusion type, 17% of the multiple type, and 7% of the arm type. As the HC were quite densely packed, the arm type was less common than in other species (see also Section 3.7). 3.3. Reconstruction of individual HC For the quantitative analysis, 99 individual HC were reconstructed together with their nerve terminals. Fig. 2 shows a schematic drawing of the emu's BP and reconstructions of individual representative HC at the ¢ve analyzed positions. Although apical HC are generally taller than basal ones, and neural ones are taller than abneural ones, the HC directly along the neural edge are much more similar to each other than their more medial (towards the BP's midline) neighbors. A most important functional characteristic of sensory cells is their innervation pattern. The emu has, like most other birds, on average one or two a¡erent and about one e¡erent nerve terminals per HC. Along the abneural edge, a zone of HC has only e¡erent innervation. The shape of the a¡erent synapses was knob- to ¢nger-like. Especially in the apex, larger a¡erent ¢bers formed synapses with several HC along their course. They usually did not branch, but contacted their HC en passant. E¡erent synaptic terminals were smaller and knob-like on neural HC and larger on abneural HC although large cup-like terminals were not common. Branching of e¡erent nerve ¢bers was often observed in the vicinity of the HC.
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Fig. 2. Schematic drawing of the emu's BP and reconstructions of individual representative HC at ¢ve positions. For each position (indicated on the papilla's outline), one HC at the neural edge (left), for the neural third (middle left), the medial third (middle right) and for the abneural third (right) is shown. Top is apical, bottom is basal, the left side is neural, the right side is abneural. Dotted terminals with thick outlines are e¡erent, unshaded terminals with thin outlines are a¡erent.
3.4. HC height and shape In the emu, HC height (Fig. 3a,b) changed gradually from the neural to the abneural edge of the basilar papilla. This was true for all ¢ve positions. Neural
HC were taller than abneural ones, and apical HC were taller than basal ones. An exception were the most neural HC: they tended to be shorter, and more similar to each other along the whole length of the papilla, compared to their more medial neighbors. This general pat-
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Fig. 3. HC morphology. On the left side, the parameter HC length (a) and HC length/width ratio (c) are shown across the papilla for the ¢ve positions, measured as a function of the distance from the neural border of the BP. Original values for single HC (symbols, for three positions to show the variation of data) and ¢fth-order polynomial least-square regressions (lines) are shown. 9% is the most basal position, 89% the most apical position studied. On the right side, the pattern of HC length (b) and HC length/width ratio (d) along the BP are shown, measured as a function of the distance from the base of the papilla. Actual values for single most neural HC (i.e. the HC directly at the neural edge), and averaged values for neural, medial and abneural HC are shown. Lines and symbols on the left side of the ¢gure: thin dotted line, and open rhomboids: 9%; thin solid line: 27%; medium solid line, and triangles: 41%; thick dashed line: 68%; thick solid line, and open circles: 89%. Lines and symbols on the right side of the ¢gure: medium solid line with ¢lled rhomboids: abneural HC; medium dashed line with ¢lled boxes: medial HC; medium solid line with open boxes: neural HC; thin solid line with open rhomboids: the most neurally situated row of HC.
Fig. 4. Maximal stereovillar height across the BP (a) and along the BP (b). Lines and symbols as in Fig. 3, with the exception that plain trend lines are given in a. Apart from the basal-apical gradient, there was also some tendency of a neural-abneural increase in stereovillar height (R2 9% = 0.69; R2 26% = 0.423; R2 41% = 0.437; R2 68% = 0.36; R2 89% = 0.354).
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tern was similar to that found in other avian species. The emu had, however, very tall HC in the apical half of its papilla, reaching up to 40 Wm. The largest di¡erence in HC height across the papilla's width was between 20 and 40% from neural. The pattern for the HC shape factor (the HC height/ width ratio, originally introduced by Takasaka and Smith (1971) to distinguish di¡erent HC types in birds) was similar, as the width of the emu HC changed much less than their height (Fig. 3c,d). There was a continuous increase of the HC height/width ratio towards the apex. Except for the most neural HC, neurally situated HC had higher values than abneural HC. The di¡erence was largest in the apical half of the papilla. At the abneural edge, the shape ratio was more constant along the length of the basilar papilla. HC with a shape ratio 6 1 were rare in the emu and occurred only in the basal third near the abneural edge. The values were, however, near 1. HC with a ratio of 0.5 or less, as found in the other examined species, were not present in the emu. 3.5. Stereovillar height As in other birds, the height of the tallest stereovilli in the bundle of the HC also increased regularly from 3^4 Wm in the papilla's base to 9^10 Wm in the apex
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(Fig. 4a,b). In contrast to the HC shape, the parameter `stereovillar height' had quite a linear pattern. The most neural HC had stereovilli of equal height to their more medial neighbors. Although the number of the HC in which the height of the tallest stereovilli could be exactly measured was small, there was a tendency in two individuals studied for abneural HC to have slightly longer stereovilli than neural ones. In the third one many of the stereovillar bundles were bent too much and exact measurements were therefore not possible. 3.6. Dimensions of the cuticular plate The height of the cuticular plate was quite constant over the width (Fig. 5a) and along the length (Fig. 5b) of the BP in the emu. It was near 3 Wm in all parts of the BP. In contrast, the width of the cuticular plate in the emu varied with the HC's apical dimensions and ranged from 3 Wm at the neural edge to 8^11 Wm in the abneural half of the BP (Fig. 5c,d). The widest cuticular plates were found in abneural and medial HC at about 40% from the BP's base. 3.7. A¡erent innervation In the emu, neural HC along the whole length of the
Fig. 5. Height (a, b) and width (c, d) of the cuticular plate across (left side) and along (right side) the BP. Lines and symbols as in Fig. 3.
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Fig. 6. HC a¡erent innervation pattern across (left side) and along (right side) the BP, shown in a similar way as in Fig. 3. a, b: Number of a¡erent terminals per HC. c, d: A¡erent contact area per HC.
BP all had a¡erent ¢bers, mostly one to three (Fig. 6a,b). The number decreased to zero towards the abneural edge of the BP. Along the abneural edge of the BP there was a zone of HC that consistently lacked a¡erent innervation. In the emu, this zone occupied the abneural 20%, and was thus narrower than in the other species studied. It was obvious that the most neural HC had the highest innervation density (up to four terminals/HC). Along the whole abneural edge of the BP, the number of a¡erent terminals was, on average, near zero, as it is also the case in other birds like the starling and the barn owl. From the values shown in Figs. 1b and 6a, we estimated that about 20% of the HC in the BP of the emu had no a¡erent innervation. The patterns of the a¡erent contact area per HC were similar (Fig. 6c,d). The area also decreased from the neural to the abneural side of the papilla. At the apex (89% position), a second zone with large a¡erent contact areas was present in the middle part of the BP, at about 40% from neural. The zone of HC without a¡erent innervation along the abneural edge of the basilar papilla was even more prominent. Along the whole abneural edge, virtually no a¡erent innervation was present.
A¡erent nerve ¢bers (that generally have no myelin sheet inside the BP) within the emu's BP were remarkably thick. In the basal region, they had a diameter up to 2 Wm, in the apical region, they even reached 7 Wm. These were the largest diameters of a¡erent papillar nerve ¢bers in the inner ear of all avian species studied
Fig. 7. Proportion of HC with non-exclusive a¡erents and of HC with contacts to neighboring HC, as a function of position along the BP.
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Fig. 8. HC e¡erent innervation pattern across (left side) and along (right side) the BP, shown in a similar way as in Fig. 3. a, b: Number of efferent terminals per HC. c, d: E¡erent contact area per HC. In a, only regression lines for the two apical positions are shown. For the other positions, such regression lines cannot be drawn.
in such detail. The a¡erent nerve ¢bers run directly from the habenula perforata to contact their HC. Only in the basal 30% of the BP all a¡erents were exclusive, i.e. they contacted only one HC. Towards the apex, there was a large proportion of a¡erent nerve ¢bers that contacted several HC (Fig. 7). Another feature in the BP were the contacts between neighboring HC mentioned above (Fig. 7). These contacts may even be true fusions that couple HC electrically. No HC-HC contacts were present in the basal 30%. HC-HC contacts were found mostly in the neural 20% of the BP, except in the most apical position where they occurred up to 40% from the neural edge. Nonexclusive a¡erent innervation, on the other hand, was frequent along the neural edge, but also present up to 60% across the papilla's width in the two apical positions (68% and 89% from the BP's base). 3.8. E¡erent innervation In general, all HC in the emu's BP had at least one e¡erent synapse. Only in the apex (68% and 89% positions, Fig. 8a), scattered HC without e¡erent nerve contacts were found, mainly in the medial part of the BP.
There was no substantial di¡erence in the number of e¡erent terminals between neural and abneural HC at any position along the BP (Fig. 8b). The e¡erent contact area per HC, however, showed an obvious increase towards the abneural side of the papilla (Fig. 8c,d). Abneural HC thus had a larger e¡erent contact area than neural HC along the whole length of the papilla, even though the number of e¡erent terminals was equal. 4. Discussion Is the inner ear of the emu primitive, average, or specialized ? The answer, generally speaking, is: the emu is a rather average bird in many respects, as far as its inner ear is concerned. The patterns in hair cell morphology and innervation in the emu closely resemble those in other bird species. This means there is a general pattern in the structures of the avian basilar papilla, and this scheme was established early in the evolution of birds. In detail, however, there are a number of signs of a more primitive character; some other features may be adaptive specializations.
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Fig. 9. HC morphology and innervation, as a function of the characteristic frequency according to the HC position on the BP. Lines and symbols as in Fig. 3b. a: HC height. b: Maximal stereovillar height. c: Number of a¡erent terminals per HC. d: A¡erent contact area per HC. e: Proportion of HC with non-exclusive a¡erents and of HC with contacts to neighboring HC. f: E¡erent contact area per HC.
The relatively large size of the BP in the emu is probably due to the larger body size of this species. Unspecialized birds, like the chicken, have similar dimensions, whereas song birds have a much smaller BP (Gleich et al., 1994). On the other hand, the HC in the basilar papilla of the emu lie very close together and they are more densely packed than in other birds. The calculation of 16 500 and 16 900 HC in the studied papillae closely resembles the number of HC counts in the SEM of about 17 000 HC given by Gleich and Manley
(1998) ; there are thus many more HC than in other avian species except for the barn owl that has a much longer BP. There are only 9300 a¡erent nerve ¢bers in a typical emu papilla (Gleich and Manley, 1998). The innervation density is thus lower than in other birds studied so far. Song birds and the highly specialized barn owl have up to twice as many a¡erent nerve ¢bers as they have HC (Fischer, 1994a; Gleich and Manley, 1998). In the BP of the emu, the HC are comparatively tall.
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Fig. 10. A comparison of HC height (a) and stereovillar height (b) in the emu and the barn owl, as a function of the characteristic frequency. The barn owl data are derived from Fischer, 1994a.
It is not quite clear whether this is a primitive condition and/or, at least partly, a specialization of the emu, as the dominant frequencies in the emu's vocalizations are below 0.5 kHz (Marchant and Higgins, 1990). The parameter `HC height' has come into discussion because its absolute value seems to be correlated to the best frequency in mammalian OHC (Pujol, 1991; Dannhof et al., 1991). As the length of the HC in birds is also correlated to the characteristic frequency (Fischer, 1994b), we postulated that the low frequencies are well represented in the emu papilla (Fischer, 1994c). In fact, Koëppl and Manley (1997) and Manley et al. (1997) have shown a good low-frequency representation in the emu BP. The range of CFs of auditory nerve units (0.04^4 kHz, Manley et al., 1997), however, corresponds well to the typical hearing range of other birds. The frequency map of the emu is a exponential function (Koëppl and Manley, 1997). In the emu, there is no strict linear correlation between the characteristic frequency and HC morphology (Fig. 9a), with the exception of stereovillar height (Fig. 9b). The height of
the tallest stereovillar row in the HC bundles seems to be another parameter that correlates with the characteristic frequency (Fischer, 1994a). In the SEM study of another ratite bird, the common rhea (Rhea americana), the values and the basal-apical increase in height were similar (JÖrgensen and Christensen, 1989). In comparison with a high-frequency specialist, the barn owl, the data on HC and stereovillar height (Fig. 10) match quite well to the corresponding frequencies. Stereovillar height as well as HC height are probably important factors in frequency analysis along the basilar papilla. The height of the cuticular plate is quite constant over the width and along the length of the BP in the emu. The same was found previously for the starling (Fischer et al., 1992), whereas in the chicken, basal HC had higher cuticular plates than apical ones (Brix et al., 1994). In contrast, the width of the cuticular plate increased in the emu with the HC's apical dimensions from the neural edge to the midline of the BP. In the chicken and the starling, similar patterns for the width of the cuticular plate were found (Brix et al., 1994;
Fig. 11. Distribution of hair cell types (THC and SHC) on the BP according to our proposed functional de¢nition (HC with a¡erent innervation (THC) and without a¡erent innervation (SHC)). The upper side is abneural, the left side is basal, the right side is apical. The neural edge is represented by the abscissa.
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Fischer et al., 1992). In the barn owl, there was an increase in the height of the cuticular plate from the base to the apex, and an increase in width from the neural to the abneural side, with the exception of the extreme base of the BP (unpublished observations). The shape of the cuticular plate seems to follow the shape of the HC. The emu's audiogram is most sensitive around 1 kHz (Manley et al., 1997). In this part of the BP, the a¡erent innervation (both number of terminals and contact area) in the most neural HC showed a peak (Fig. 9c,d) in all three BP studied. This corresponds well to the results in the other analyzed species, the chicken, starling, and barn owl that also have a maximum in a¡erent innervation in the frequency range where they hear most sensitively (Fischer, 1994b). For the e¡erent innervation (Fig. 9f), such a correlation is also not found in any of the other studied species (Fischer, 1994b). There is, on average, one e¡erent terminal per HC in all avian species studied so far. Along the BP, abneural HC in the middle part often had larger e¡erent contact areas in the studied species although typical cup-like synapses were rare. The number of a¡erent nerve terminals per HC is typically low in birds. In the emu, neural HC along the whole length of the BP all had a¡erent ¢bers, mostly one to three. In birds, most HC a¡erents are exclusive, i.e. one ¢ber innervates only one HC. In the BP of the emu this was only true for the basal 30% of the BP. Especially in its apex, many HC had non-exclusive a¡erent innervation, i.e. one a¡erent ¢ber contacted several neighboring HC. Koëppl and Manley (1997) obtained similar results by iontophoretically labeling primary a¡erents. In parallel with this high proportion of non-exclusive a¡erents, there are numerous contacts between neighboring HC. Thus, there appears to be a high degree of peripheral integration in the apex. These non-exclusive a¡erents and HC-HC contacts in a large proportion of the emu papilla seem to be a primitive feature. In the emu BP, this is restricted to a¡erents with best frequencies below 1 kHz (Fig. 9e). In other, and even in most specialized species like the barn owl, a comparable situation is found only in the most apical part of their papillae. The apex is considered to be the evolutionary oldest portion of the avian basilar papilla (Smith et al., 1985; Lavigne-Rebillard et al., 1985). In modern birds, like the song birds, the relative number of a¡erents has increased and thus the innervation density of the THC (for review see Gleich and Manley, 1998). This was much more the case in the high-frequency base of the avian papilla, and is especially elaborate in a high-frequency specialist, the barn owl, where the most neural HC may have up to 20 a¡erent terminals and thus reach the density of a¡erent innervation of mammalian IHC (Fischer, 1994a).
The majority of HC receive a¡erent innervation. The zone of the true SHC (HC without a¡erents, Fischer, 1994a,b) along the abneural edge of the papilla is rather narrow in the emu, and this is probably a primitive condition (Fig. 11). In species specialized for a higher frequency range, like the barn owl, the SHC are the dominating HC type in the BP's base. SHC obviously are important elements in higher-frequency hearing. However, a zone without a¡erent innervation is also present in the primitive emu, and this fact is corroborated by the failure to label primary a¡erents near the abneural edge of the emu BP (Koëppl and Manley, 1997). This points to a unique and common characteristic of the avian hearing epithelium. As far as we know, such avian abneural HC are the only sensory cells known to routinely lack a¡erent innervation. This o¡ered the possibility to clearly (and functionally) de¢ne SHC as those HC without a¡erent but with efferent innervation, and THC as those HC with a¡erent (and mostly also e¡erent) innervation (Fischer, 1994a). On the other hand, the `standard bird', the chicken, has, in its apical part of the BP, many abneural HC with a¡erent terminals. It is not clear whether this is rather a primitive state or an e¡ect of domestication. Di¡erent results, e.g. on the pattern of the size of e¡erent terminals (a gradient, or a rather sudden change in size) across the BP (Ofsie and Cotanche, 1996 ; Zidanic and Fuchs, 1996 ; Fischer, 1992), point to some variation between domesticated chickens. One should be cautious in considering the chicken BP as `the typical avian BP'. The THC are more similar to typical HC of more primitive vertebrate groups (Takasaka and Smith, 1971 ; Chandler, 1984). They perform the original sensory function, i.e. processing information and transmitting it towards the brain (Manley et al., 1989; Gleich, 1989 ; Manley and Gleich, 1992). A large proportion of the THC (and especially the most neural ones which have the largest a¡erent innervation) lie over the cartilage-like neural limbus, and not over the free (movable) basilar membrane. For this reason, the tectorial membrane must play a crucial role in the mechanical stimulation of the THC. Like the OHC in mammals, a derived hair cell type has evolved in birds (SHC); they always lie over the basilar membrane. As the function of the SHC cannot be sensory (in contrast to the mammalian OHC), they probably are e¡ectors that can be in£uenced by their e¡erents. They possibly change the mechanics of the tectorial membrane, e.g. by a change in sti¡ness or by bundle movements, and they also could modify the stimulus on the THC ; in this way, the SHC would play a functionally supportive role, as it is also proposed for the OHC in mammals (see e.g. Ashmore, 1994). The zone of hyaline cells along the abneural edge of the papilla is very broad in the emu. These hyaline cells
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were studied in detail in the chicken ; they contain actin and myosin in a regular array (Cotanche et al., 1992). They receive en passant innervation from the e¡erents that enter the basilar papilla through the habenula perforata (Oesterle et al., 1992). After contacting the hyaline cells, e¡erent ¢bers turn back and innervate the SHC (Zidanic and Fuchs, 1996). Therefore it is reasonable to assume that the SHC and the hyaline cells together form a functionally supporting system in the avian BP. In the emu, the hyaline cells are prominent while the SHC zone is rather small. Detailed analyses of the SHC/hyaline cell system are needed to obtain a better knowledge of this supposed supporting apparatus in the avian cochlea. Acknowledgments I am very grateful to O. Gleich and C. Koëppl for the ¢xed and embedded specimens. Many thanks also to C. Koëppl, H.-J. Leppelsack and G.A. Manley for commenting on an earlier version of this paper. I especially thank G.A. Manley for his support and the correction of my English. The photographic work of Gaby Schwabedissen is gratefully acknowledged. The Deutsche Forschungsgemeinschaft (DFG) supported this and other comparative studies with a grant to G.A. Manley within the SFB 204 (`Gehoër'). Many thanks are also due to three anonymous reviewers for their constructive comments.
References Ashmore, J.F., 1994. The cellular machinery of the cochlea. Exp. Physiol. 79, 113^134. Brix, J., Fischer, F.P., Manley, G.A., 1994. The cuticular plate of the hair cell in relation to morphological gradients of the chicken basilar papilla. Hear. Res. 75, 244^256. Carrol, R.L., 1988. Vertebrate Palaeontology and Evolution. Freeman, New York. Chandler, J.P., 1984. Light and electron microscopic studies of the basilar papilla in the duck, Anas platyrhynchos: I. The hatchling. J. Comp. Neurol. 222, 506^522. Chen, L., Salvi, R., Shero, M., 1994. Cochlear frequency-place map in adult chickens: Intracellular biocytin labeling. Hear. Res. 81, 130^ 136. Code, R.A., Carr, C.E., 1994. Choline acetyltransferase-immunoreactive cochlear e¡erent neurons in the chick auditory brainstem. J. Comp. Neurol. 340, 161^173. Cole, K.S., Gummer, A.W., 1990. A double-label study of e¡erent projections to the cochlea of the chicken, Gallus domesticus. Exp. Brain Res. 82, 585^588. Corwin, J.T., Cotanche, D.A., 1988. Regeneration of sensory hair cells after acoustic trauma. Science 240, 1772^1774. Cotanche, D.A., 1987. Regeneration of the tectorial membrane in the chick cochlea following severe acoustic trauma. Hear. Res. 30, 197^206. Cotanche, D.A., Henson, M.M., Henson, O.W., 1992. Contractile
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proteins in the hyaline cells of the chicken cochlea. J. Comp. Neurol. 324, 353^364. Cracraft, J., 1981. Towards a phylogenetic classi¢cation of recent birds of the world (class Aves). Auk 98, 681^714. Dannhof, B.J., Roth, B., Bruns, V., 1991. Length of hair cells as a measure of frequency representation in the mammalian inner ear? Naturwissenschaften 78, 570^573. Feduccia, A., 1980. The Age of Birds. Harvard University Press, Cambridge, MA. Fischer, F.P., 1992. Quantitative analysis of the innervation of the chicken basilar papilla. Hear. Res. 61, 167^178. Fischer, F.P., 1994a. Quantitative TEM analysis of the barn owl basilar papilla. Hear. Res. 73, 1^15. Fischer, F.P., 1994b. General pattern and morphological specializations of the avian cochlea. Scan. Microsc. 8, 351^364. Fischer, F.P., 1994c. Morphology and innervation pattern of hair cells in the basilar papilla of the emu. Abstracts of the 31th Inner Ear Biology Workshop, Montpellier, 10-13 September 1994, p. 12. Fischer, F.P., Koëppl, C., Manley, G.A., 1988. The basilar papilla of the barn owl Tyto alba: A quantitative morphological SEM analysis. Hear. Res. 34, 87^101. Fischer, F.P., Brix, J., Singer, I., Miltz, C., 1991. Contacts between hair cells in the avian cochlea. Hear. Res. 53, 281^292. Fischer, F.P., Singer, I., Miltz, C., Manley, G.A., 1992. Morphological gradients in the starling basilar papilla. J. Morphol. 213, 225^ 240. Fuchs, P.A., 1992. Ionic currents in cochlear hair cells. Prog. Neurobiol. 39, 493^505. Fuchs, P.A., Murrow, B.W., 1992. Cholinergic inhibition of short (outer) hair cells of the chick's cochlea. J. Neurosci. 12 (3), 800^ 809. Gleich, O., 1989. Auditory primary a¡erents in the starling: Correlation of function and morphology. Hear. Res. 37, 255^268. Gleich, O., Manley, G.A., 1998. The Hearing Organ of Birds and their Relatives. Springer, Berlin (in press). Gleich, O., Manley, G.A., Mandl, A., Dooling, R., 1994. The basilar papilla of the canary and the zebra ¢nch: a quantitative scanning electron microscopic description. J. Morphol. 221, 1^24. Jones, S.M., Jones, T.A., 1995. The tonotopic map of the embryonic chick cochlea. Hear. Res. 82, 149^157. JÖrgensen, J.M., Christensen, J.T., 1989. The inner ear of ther common rhea (Rhea americana L.). Brain Behav. Evol. 34, 273^280. Kaiser, A., Manley, G.A., 1994. Physiology of putative single cochlear e¡erents in the chicken. J. Neurophysiol. 72, 2966^2979. Klinke, R., Smolders, J.W.T., 1993. Performance of the avian inner ear. In: Allum, J.H.J., Allum-Mecklenburg, D.J., Harris, F.P., Probst, R. (Eds.), Progress in Brain Research, Vol. 97, pp. 31^ 43. Koëppl, C., Manley, G.A., 1997. Frequency representation in the emu basilar papilla. J. Acoust. Soc. Am. 101, 1574^1584. Koëppl, C., Gleich, O., Manley, G.A., 1993. An auditory fovea in the barn owl cochlea. J. Comp. Physiol. 171, 695^704. Lavigne-Rebillard, M., Cousillas, H., Pujol, R., 1985. The very distal part of the basilar papilla in the chicken: A morphological approach. J. Comp. Neurol. 238, 340^347. Manley, G.A., 1995. The avian hearing organ: a status report. In: Manley, G.A., Klump, G.M., Koëppl, C., Fastl, H., Oeckinghaus, H. (Eds.), Advances in Hearing Research. World Scienti¢c, Singapore, pp. 219^229. Manley, G.A., Gleich, O., 1992. Evolution and specialization of function in the avian auditory periphery. In: Webster, D.B., Fay, R., Popper, A.N. (Eds.), The Evolutionary Biology of Hearing. Springer, Berlin. Manley, G.A., Brix, J., Kaiser, A., 1987. Developmental stability of the tonotopic organization of the chick's basilar papilla. Science 237, 655^656. Manley, G.A., Gleich, O., Kaiser, A., Brix, J., 1989. Functional di¡er-
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entiation of sensory cells in the avian auditory periphery. J. Comp. Physiol. A 164, 289^296. Manley, G.A., Meyer, B., Fischer, F.P., Schwabedissen, G., Gleich, O., 1996. Surface morphology of the basilar papilla of the tufted duck Aythya fuligula and the domestic chicken Gallus gallus domesticus. J. Morphol. 227, 197^212. Manley, G.A., Koëppl, C., Yates, G.K., 1997. Activity of primary auditory neurones in the cochlear ganglion of the Emu Dromaius novaehollandiae I: Spontaneous discharge, frequency tuning and phase locking. J. Acoust. Soc. Am. 101, 1560^1573. Marchant, S., Higgins, P.J., 1990. Handbook of Australian, New Zealand and Antarctic Birds. Oxford University Press, Melbourne, pp. 47^58. Oesterle, E.C., Cunningham, D.E., Rubel, E.W., 1992. Ultrastructure of hyaline, border, and vacuole cells in chick inner ear. J. Comp. Neurol. 318, 64^82. Ofsie, M.S., Cotanche, D.A., 1996. Distribution of nerve ¢bers in the basilar papilla of normal and sound-damaged chick cochleae. J. Comp. Neurol. 370, 281^294.
Pujol, R., 1991. Is the length of OHCs correlated with the frequency coding of the cochlea? Midwinter Meeting, Assoc. Res. Otolaryngol., St. Petersburg, FL, p. 125. Ryals, B.M., Rubel, E.W., 1988. Hair cell regeneration after acoustic trauma in adult Coturnix quail. Science 240, 1774^1776. Smith, C.A., Konishi, M., Schull, N., 1985. Structure of the barn owl's (Tyto alba) inner ear. Hear. Res. 17, 237^247. Smolders, J.W.T., Ding-Pfennigdor¡, D., Klinke, R., 1995. A functional map of the pigeon basilar papilla: correlation of the properties of single auditory nerve ¢bres and their peripheral origin. Hear. Res. 92, 151^169. Takasaka, T., Smith, C.A., 1971. The structure and innervation of the pigeon's basilar papilla. J. Ultrastruct. Res. 35, 20^65. Tanaka, K., Smith, C.A., 1978. Structure of the chicken's inner ear: SEM and TEM study. Am. J. Anat. 153, 251^271. Zidanic, M., Fuchs, P.A., 1996. Synapsin-like immunoreactivity in the chick-cochlea: speci¢c labeling of e¡erent nerve terminals. Audit. Neurosci. 2, 347^362.
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Hair cell morphology and innervation in the basilar papilla of the emu (Dromaius novaehollandiae) Franz Peter Fischer * Institut fuër Zoologie, Technische Universitaët Muënchen, Lichtenbergstrasse 4, D-85747 Garching, Germany Received 9 December 1997; revised 13 April 1998; accepted 18 April 1998
Abstract The emu, being a member of the rather primitive bird group of the palaeognathid Ratitae, may reveal primitives features of the avian basilar papilla. There are, however, no qualitative differences with the papillae of other birds such as the chicken or the starling. There are only quantitative differences in the continuous morphological gradients (such as hair cell height, stereovillar height) from neural to abneural, and from the base to the apex of the papilla. Only few (about two in the emu) afferent terminals and on average one efferent fiber contact each hair cell. Along the abneural edge, there is a population of hair cells that lack afferent innervation (short hair cells), suggesting that their function must lie in the papilla itself. There is thus a general pattern in the structures of the avian basilar papilla. In detail, however, a number of primitive characters were observed in the emu, as compared to advanced birds such as the starling and the barn owl. The hair cells are very densely packed and comparatively tall (up to 40 Wm in the apex). This anatomy correlates well with the good lower-frequency hearing (see Koëppl and Manley, J. Acoust. Soc. Am. 101 (1997) 1574^1584). The afferent nerve fibers contacting the hair cells within the basilar papilla are rather thick, and there are a large number of afferent fibers that contact more than one hair cell. The zone of hair cells without afferent innervation (short hair cells) along the abneural edge of the basilar papilla is rather narrow in the emu. z 1998 Elsevier Science B.V. All rights reserved. Key words: Bird; Emu; Cochlea; Hair cell; Innervation; Evolution
1. Introduction In the last decade, di¡erent research groups have shown that hair cells (HC) in the avian inner ear can regenerate, e.g. after acoustic trauma, in contrast to the situation in the mammalian cochlea (e.g. Cotanche, 1987 ; Corwin and Cotanche, 1988; Ryals and Rubel, 1988). In this new research area, the chicken has become the standard bird. The morphology, innervation and synaptic physiology of the HC in the chicken basilar papilla (BP) have been studied intensively (Cole and Gummer, 1990 ; Fischer, 1992; Ofsie and Cotanche, 1996 ; Zidanic and Fuchs, 1996; Fuchs, 1992; Fuchs and Murrow, 1992; Code and Carr, 1994; Kaiser and Manley, 1994). The ears of other avian species have been much less well investigated. * Tel.: +49 (89) 2891-3662 (3661); Fax: +49 (89) 2891-3674; E-mail: [email protected]
The inner ears of di¡erent bird species have several morphological and functional similarities, but also marked di¡erences from the mammalian organ of Corti. The avian hearing epithelium, the basilar papilla, is an elongated £at band. In contrast to the mammalian organ of Corti, there are no distinct hair cell rows like inner and outer hair cells (IHC and OHC). In birds, the hair cells occupy the whole surface, and are separated from each other by narrow supporting cells. Like mammals, birds have di¡erent forms of HC across the papilla, the extremes being the tall hair cells (THC) on the neural and the short hair cells (SHC) on the abneural side, with, however, a continuous transition in between. The mechanism of hearing is far less well understood in birds than in mammals (Klinke and Smolders, 1993; Manley et al., 1989; Manley, 1995). The comparison of anatomy and innervation pattern in the hearing organ of di¡erently specialized avian species is one approach to obtain a better insight into the relationship between
0378-5955 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 9 8 ) 0 0 0 7 2 - 0
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structure and function, between the anatomy of the hearing epithelium and its innervation on the one hand, and physiological data on the other hand. We studied, at the transmission electron microscopy (TEM) level, the patterns and gradients in HC morphology and innervation as compared to their position on the BP and thus to their characteristic frequencies. This provides the opportunity of obtaining a large and detailed body of systematic quantitative information. So far, we have studied the inner ear of the chicken (Fischer, 1992), the starling (Fischer et al., 1992) and the barn owl (Fischer, 1994a) in such detail. Frequency maps of avian BP are currently available for the chicken (Manley et al., 1987; Chen et al., 1994; Jones and Jones, 1995), the starling (Gleich, 1989), the pigeon (Klinke and Smolders, 1993; Smolders et al., 1995), the barn owl (Koëppl et al., 1993) and recently also for the emu (Koëppl and Manley, 1997). The Australian emu (Dromaius novaehollandiae) is considered to be a rather primitive bird (Feduccia, 1980 ; Carrol, 1988); it is a member of the palaeognathid line that separated early in the avian radiation from the neognathid birds. Its body structures show several primitive features (Cracraft, 1981). The study of its inner ear may possibly reveal primitive features in the morphology and innervation pattern of the avian hearing organ. Therefore we quantitatively analyzed the BP of the emu in a similar way as before in the chicken, the starling and the barn owl. 2. Materials and methods For this quantitative systematic study, ¢ve positions along the basilar papilla (BP) of a 4^5 day old (posthatch) emu (Dromaius novaehollandiae LATHAM 1790) were analyzed using serial TEM sections. Two papillae of additional individuals (a hatchling and a 70 week old emu) served as TEM controls to exclude the possibility that the analyzed specimen was abnormal. The hatchling was analyzed at ¢ve similar positions, the other one only at three positions. As the data were similar, only one BP (that of the 4^5 day old emu) is described here in detail. Some aspects of the BP of the emu hatchling were already included in a separate comparative study (Fischer, 1994b). Additional ears were studied at the light microscopic level. The emu was anesthetized with a combination of Chlorthesin and Nembutal, and electrical recordings were made from the acoustic nerve for several hours (Manley et al., 1997). Then the round window was opened and the columella removed. A small volume (a few ml) of cold ¢xative (5% glutaraldehyde in phosphate bu¡er, pH 7.4) was slowly introduced through the oval window and drawn out of the ear via the round window. The animal was then killed by an over-
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dose of anesthetic, and the ear was dissected free and placed overnight in chilled ¢xative. Then it was washed in chilled bu¡er, and post¢xed in 2% osmium tetroxide in bu¡er for 2 h. After dehydration in ethanol it was embedded in Araldit. The ¢xation protocol for the other ears was slightly di¡erent, but the ultrastructure and dimensions of BP and HC were very similar. Serial semithin and ultrathin sections were cut with a Reichert ultramicrotome. The semithin sections were stained with 1% toluidine blue in 1% borax solution, the ultrathin sections with uranyl acetate and lead citrate. The ultrathin series were at the positions 9%, 26%, 41%, 68% and 89% as measured from the base of the BP. This corresponds to frequencies of 3.3, 1.14, 0.7, 0.2 and 0.08 kHz, respectively (Koëppl and Manley, 1997). The sections were studied in a Jeol JEM 100 CX TEM at a primary magni¢cation of 2600U and every fourth section was photographed. In every session, an exact calibration of the TEM was carried out. The photographs were subsequently enlarged to 5000U. Details were studied at higher magni¢cation. All measurements given in this paper are for the ¢xed and embedded specimen, without correction for shrinkage (as previously in the other species). The HC and their nerve endings were drawn on transparent sheets. These were placed on top of each other, and, with the HC surface and the nucleus as landmarks, the sheets were used for the reconstructions. As the HC in the apex of the BP are slightly obliquely orientated and also often twisted, exact measurements of HC length were only possible from complete reconstructions from serial sections of known thickness. The numbers of nerve terminals were directly derived from these reconstructions. Synaptic areas were calculated from the length of contact zones and the thickness of the sections. Altogether, 107 HC and their innervation pattern were reconstructed (14 HC at the 9% position, 16 at the 27% position, 24 at the 41% position, 23 at the 68% position, and 30 at the 89% position). Ninety-nine HC could be used for the analysis of the innervation pattern. All procedures were in conformity with the Australian regulations for animal protection and were approved by the Animal Experimentation Ethics Committee of the University of Western Australia (64/93/93 and 180/94/94). 2.1. De¢nitions To avoid confusion, in this paper the three terms length, width and height are used as follows : `length' is measured parallel to the BP's length, `width' is measured parallel to the BP's width, and `height' is measured roughly perpendicular to both (e.g., along the HC's long axis).
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Fig. 1. Dimensions of the BP (a) and number of HC across the BP (b). Data points and smoothened lines. The abscissa represents the neural border of the BP.
3. Results 3.1. Dimensions of the emu basilar papilla Compared with most other birds (a marked exception being the barn owl; Smith et al., 1985; Fischer et al., 1988 ; Gleich et al., 1994; Manley et al., 1996), the dimensions of the emu's BP were in the upper range. The length of the BP was 4.65 mm (¢xed and embedded). The width ranged from about 80 Wm in the base to 463 Wm near the apical end (Fig. 1a). The number of HC across the BP was 13 in the base and 49 in the broadest part (near the apex). Taking the number of HC across the BP, the HC diameters, and the BP dimensions at the ¢ve positions into account, an overall number of about 16 500 hair cells was calculated for the 4^5 day old emu and 16 900 for the hatchling. A substantial proportion of the HC were placed over the neural limbus (Fig. 1b). The zone of the hyaline cells along the abneural border was remarkably broad and occupied up to 100 Wm of the width near the apex (Fig. 1a). 3.2. HC morphology along and across the BP Basal and apical HC had quite a di¡erent shape. Apical HC were tall, basal HC were much shorter. Apical HC had much active cytoplasm (cytoplasm without the cuticular plate), whereas basal HC generally had less active cytoplasm, especially those on the abneural side of the papilla. Over the width of the BP, taller HC on the neural side graded into shorter HC on the abneural side. Compared to other avian species, the HC in the emu were remarkably tall. Their width, however, was average. They were unusually densely packed, with only little space for the separating supporting cells, especially in the apical half of the BP, where the HC virtually touched each other. The ultrastructure of the HC and their synapses was similar to that in other avian species (e.g. chicken : Tanaka and Smith, 1978; Fischer, 1992; pigeon : Takasa-
ka and Smith, 1971 ; starling : Fischer et al., 1992; duck: Chandler, 1984) and will therefore not be repeated here in detail. Kinocilia were occasionally observed throughout the whole BP. Where they were vestigial, a basal body remained. As in the other avian species examined, contacts between neighboring HC (Fischer et al., 1991) were frequent in the apical half of the BP (see also Fig. 7). All four previously described contact types were found, including true membrane fusions. Altogether, of 34 contacts evaluated, 53% were of the touch type, 23% of the fusion type, 17% of the multiple type, and 7% of the arm type. As the HC were quite densely packed, the arm type was less common than in other species (see also Section 3.7). 3.3. Reconstruction of individual HC For the quantitative analysis, 99 individual HC were reconstructed together with their nerve terminals. Fig. 2 shows a schematic drawing of the emu's BP and reconstructions of individual representative HC at the ¢ve analyzed positions. Although apical HC are generally taller than basal ones, and neural ones are taller than abneural ones, the HC directly along the neural edge are much more similar to each other than their more medial (towards the BP's midline) neighbors. A most important functional characteristic of sensory cells is their innervation pattern. The emu has, like most other birds, on average one or two a¡erent and about one e¡erent nerve terminals per HC. Along the abneural edge, a zone of HC has only e¡erent innervation. The shape of the a¡erent synapses was knob- to ¢nger-like. Especially in the apex, larger a¡erent ¢bers formed synapses with several HC along their course. They usually did not branch, but contacted their HC en passant. E¡erent synaptic terminals were smaller and knob-like on neural HC and larger on abneural HC although large cup-like terminals were not common. Branching of e¡erent nerve ¢bers was often observed in the vicinity of the HC.
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Fig. 2. Schematic drawing of the emu's BP and reconstructions of individual representative HC at ¢ve positions. For each position (indicated on the papilla's outline), one HC at the neural edge (left), for the neural third (middle left), the medial third (middle right) and for the abneural third (right) is shown. Top is apical, bottom is basal, the left side is neural, the right side is abneural. Dotted terminals with thick outlines are e¡erent, unshaded terminals with thin outlines are a¡erent.
3.4. HC height and shape In the emu, HC height (Fig. 3a,b) changed gradually from the neural to the abneural edge of the basilar papilla. This was true for all ¢ve positions. Neural
HC were taller than abneural ones, and apical HC were taller than basal ones. An exception were the most neural HC: they tended to be shorter, and more similar to each other along the whole length of the papilla, compared to their more medial neighbors. This general pat-
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Fig. 3. HC morphology. On the left side, the parameter HC length (a) and HC length/width ratio (c) are shown across the papilla for the ¢ve positions, measured as a function of the distance from the neural border of the BP. Original values for single HC (symbols, for three positions to show the variation of data) and ¢fth-order polynomial least-square regressions (lines) are shown. 9% is the most basal position, 89% the most apical position studied. On the right side, the pattern of HC length (b) and HC length/width ratio (d) along the BP are shown, measured as a function of the distance from the base of the papilla. Actual values for single most neural HC (i.e. the HC directly at the neural edge), and averaged values for neural, medial and abneural HC are shown. Lines and symbols on the left side of the ¢gure: thin dotted line, and open rhomboids: 9%; thin solid line: 27%; medium solid line, and triangles: 41%; thick dashed line: 68%; thick solid line, and open circles: 89%. Lines and symbols on the right side of the ¢gure: medium solid line with ¢lled rhomboids: abneural HC; medium dashed line with ¢lled boxes: medial HC; medium solid line with open boxes: neural HC; thin solid line with open rhomboids: the most neurally situated row of HC.
Fig. 4. Maximal stereovillar height across the BP (a) and along the BP (b). Lines and symbols as in Fig. 3, with the exception that plain trend lines are given in a. Apart from the basal-apical gradient, there was also some tendency of a neural-abneural increase in stereovillar height (R2 9% = 0.69; R2 26% = 0.423; R2 41% = 0.437; R2 68% = 0.36; R2 89% = 0.354).
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tern was similar to that found in other avian species. The emu had, however, very tall HC in the apical half of its papilla, reaching up to 40 Wm. The largest di¡erence in HC height across the papilla's width was between 20 and 40% from neural. The pattern for the HC shape factor (the HC height/ width ratio, originally introduced by Takasaka and Smith (1971) to distinguish di¡erent HC types in birds) was similar, as the width of the emu HC changed much less than their height (Fig. 3c,d). There was a continuous increase of the HC height/width ratio towards the apex. Except for the most neural HC, neurally situated HC had higher values than abneural HC. The di¡erence was largest in the apical half of the papilla. At the abneural edge, the shape ratio was more constant along the length of the basilar papilla. HC with a shape ratio 6 1 were rare in the emu and occurred only in the basal third near the abneural edge. The values were, however, near 1. HC with a ratio of 0.5 or less, as found in the other examined species, were not present in the emu. 3.5. Stereovillar height As in other birds, the height of the tallest stereovilli in the bundle of the HC also increased regularly from 3^4 Wm in the papilla's base to 9^10 Wm in the apex
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(Fig. 4a,b). In contrast to the HC shape, the parameter `stereovillar height' had quite a linear pattern. The most neural HC had stereovilli of equal height to their more medial neighbors. Although the number of the HC in which the height of the tallest stereovilli could be exactly measured was small, there was a tendency in two individuals studied for abneural HC to have slightly longer stereovilli than neural ones. In the third one many of the stereovillar bundles were bent too much and exact measurements were therefore not possible. 3.6. Dimensions of the cuticular plate The height of the cuticular plate was quite constant over the width (Fig. 5a) and along the length (Fig. 5b) of the BP in the emu. It was near 3 Wm in all parts of the BP. In contrast, the width of the cuticular plate in the emu varied with the HC's apical dimensions and ranged from 3 Wm at the neural edge to 8^11 Wm in the abneural half of the BP (Fig. 5c,d). The widest cuticular plates were found in abneural and medial HC at about 40% from the BP's base. 3.7. A¡erent innervation In the emu, neural HC along the whole length of the
Fig. 5. Height (a, b) and width (c, d) of the cuticular plate across (left side) and along (right side) the BP. Lines and symbols as in Fig. 3.
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Fig. 6. HC a¡erent innervation pattern across (left side) and along (right side) the BP, shown in a similar way as in Fig. 3. a, b: Number of a¡erent terminals per HC. c, d: A¡erent contact area per HC.
BP all had a¡erent ¢bers, mostly one to three (Fig. 6a,b). The number decreased to zero towards the abneural edge of the BP. Along the abneural edge of the BP there was a zone of HC that consistently lacked a¡erent innervation. In the emu, this zone occupied the abneural 20%, and was thus narrower than in the other species studied. It was obvious that the most neural HC had the highest innervation density (up to four terminals/HC). Along the whole abneural edge of the BP, the number of a¡erent terminals was, on average, near zero, as it is also the case in other birds like the starling and the barn owl. From the values shown in Figs. 1b and 6a, we estimated that about 20% of the HC in the BP of the emu had no a¡erent innervation. The patterns of the a¡erent contact area per HC were similar (Fig. 6c,d). The area also decreased from the neural to the abneural side of the papilla. At the apex (89% position), a second zone with large a¡erent contact areas was present in the middle part of the BP, at about 40% from neural. The zone of HC without a¡erent innervation along the abneural edge of the basilar papilla was even more prominent. Along the whole abneural edge, virtually no a¡erent innervation was present.
A¡erent nerve ¢bers (that generally have no myelin sheet inside the BP) within the emu's BP were remarkably thick. In the basal region, they had a diameter up to 2 Wm, in the apical region, they even reached 7 Wm. These were the largest diameters of a¡erent papillar nerve ¢bers in the inner ear of all avian species studied
Fig. 7. Proportion of HC with non-exclusive a¡erents and of HC with contacts to neighboring HC, as a function of position along the BP.
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Fig. 8. HC e¡erent innervation pattern across (left side) and along (right side) the BP, shown in a similar way as in Fig. 3. a, b: Number of efferent terminals per HC. c, d: E¡erent contact area per HC. In a, only regression lines for the two apical positions are shown. For the other positions, such regression lines cannot be drawn.
in such detail. The a¡erent nerve ¢bers run directly from the habenula perforata to contact their HC. Only in the basal 30% of the BP all a¡erents were exclusive, i.e. they contacted only one HC. Towards the apex, there was a large proportion of a¡erent nerve ¢bers that contacted several HC (Fig. 7). Another feature in the BP were the contacts between neighboring HC mentioned above (Fig. 7). These contacts may even be true fusions that couple HC electrically. No HC-HC contacts were present in the basal 30%. HC-HC contacts were found mostly in the neural 20% of the BP, except in the most apical position where they occurred up to 40% from the neural edge. Nonexclusive a¡erent innervation, on the other hand, was frequent along the neural edge, but also present up to 60% across the papilla's width in the two apical positions (68% and 89% from the BP's base). 3.8. E¡erent innervation In general, all HC in the emu's BP had at least one e¡erent synapse. Only in the apex (68% and 89% positions, Fig. 8a), scattered HC without e¡erent nerve contacts were found, mainly in the medial part of the BP.
There was no substantial di¡erence in the number of e¡erent terminals between neural and abneural HC at any position along the BP (Fig. 8b). The e¡erent contact area per HC, however, showed an obvious increase towards the abneural side of the papilla (Fig. 8c,d). Abneural HC thus had a larger e¡erent contact area than neural HC along the whole length of the papilla, even though the number of e¡erent terminals was equal. 4. Discussion Is the inner ear of the emu primitive, average, or specialized ? The answer, generally speaking, is: the emu is a rather average bird in many respects, as far as its inner ear is concerned. The patterns in hair cell morphology and innervation in the emu closely resemble those in other bird species. This means there is a general pattern in the structures of the avian basilar papilla, and this scheme was established early in the evolution of birds. In detail, however, there are a number of signs of a more primitive character; some other features may be adaptive specializations.
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Fig. 9. HC morphology and innervation, as a function of the characteristic frequency according to the HC position on the BP. Lines and symbols as in Fig. 3b. a: HC height. b: Maximal stereovillar height. c: Number of a¡erent terminals per HC. d: A¡erent contact area per HC. e: Proportion of HC with non-exclusive a¡erents and of HC with contacts to neighboring HC. f: E¡erent contact area per HC.
The relatively large size of the BP in the emu is probably due to the larger body size of this species. Unspecialized birds, like the chicken, have similar dimensions, whereas song birds have a much smaller BP (Gleich et al., 1994). On the other hand, the HC in the basilar papilla of the emu lie very close together and they are more densely packed than in other birds. The calculation of 16 500 and 16 900 HC in the studied papillae closely resembles the number of HC counts in the SEM of about 17 000 HC given by Gleich and Manley
(1998) ; there are thus many more HC than in other avian species except for the barn owl that has a much longer BP. There are only 9300 a¡erent nerve ¢bers in a typical emu papilla (Gleich and Manley, 1998). The innervation density is thus lower than in other birds studied so far. Song birds and the highly specialized barn owl have up to twice as many a¡erent nerve ¢bers as they have HC (Fischer, 1994a; Gleich and Manley, 1998). In the BP of the emu, the HC are comparatively tall.
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Fig. 10. A comparison of HC height (a) and stereovillar height (b) in the emu and the barn owl, as a function of the characteristic frequency. The barn owl data are derived from Fischer, 1994a.
It is not quite clear whether this is a primitive condition and/or, at least partly, a specialization of the emu, as the dominant frequencies in the emu's vocalizations are below 0.5 kHz (Marchant and Higgins, 1990). The parameter `HC height' has come into discussion because its absolute value seems to be correlated to the best frequency in mammalian OHC (Pujol, 1991; Dannhof et al., 1991). As the length of the HC in birds is also correlated to the characteristic frequency (Fischer, 1994b), we postulated that the low frequencies are well represented in the emu papilla (Fischer, 1994c). In fact, Koëppl and Manley (1997) and Manley et al. (1997) have shown a good low-frequency representation in the emu BP. The range of CFs of auditory nerve units (0.04^4 kHz, Manley et al., 1997), however, corresponds well to the typical hearing range of other birds. The frequency map of the emu is a exponential function (Koëppl and Manley, 1997). In the emu, there is no strict linear correlation between the characteristic frequency and HC morphology (Fig. 9a), with the exception of stereovillar height (Fig. 9b). The height of
the tallest stereovillar row in the HC bundles seems to be another parameter that correlates with the characteristic frequency (Fischer, 1994a). In the SEM study of another ratite bird, the common rhea (Rhea americana), the values and the basal-apical increase in height were similar (JÖrgensen and Christensen, 1989). In comparison with a high-frequency specialist, the barn owl, the data on HC and stereovillar height (Fig. 10) match quite well to the corresponding frequencies. Stereovillar height as well as HC height are probably important factors in frequency analysis along the basilar papilla. The height of the cuticular plate is quite constant over the width and along the length of the BP in the emu. The same was found previously for the starling (Fischer et al., 1992), whereas in the chicken, basal HC had higher cuticular plates than apical ones (Brix et al., 1994). In contrast, the width of the cuticular plate increased in the emu with the HC's apical dimensions from the neural edge to the midline of the BP. In the chicken and the starling, similar patterns for the width of the cuticular plate were found (Brix et al., 1994;
Fig. 11. Distribution of hair cell types (THC and SHC) on the BP according to our proposed functional de¢nition (HC with a¡erent innervation (THC) and without a¡erent innervation (SHC)). The upper side is abneural, the left side is basal, the right side is apical. The neural edge is represented by the abscissa.
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Fischer et al., 1992). In the barn owl, there was an increase in the height of the cuticular plate from the base to the apex, and an increase in width from the neural to the abneural side, with the exception of the extreme base of the BP (unpublished observations). The shape of the cuticular plate seems to follow the shape of the HC. The emu's audiogram is most sensitive around 1 kHz (Manley et al., 1997). In this part of the BP, the a¡erent innervation (both number of terminals and contact area) in the most neural HC showed a peak (Fig. 9c,d) in all three BP studied. This corresponds well to the results in the other analyzed species, the chicken, starling, and barn owl that also have a maximum in a¡erent innervation in the frequency range where they hear most sensitively (Fischer, 1994b). For the e¡erent innervation (Fig. 9f), such a correlation is also not found in any of the other studied species (Fischer, 1994b). There is, on average, one e¡erent terminal per HC in all avian species studied so far. Along the BP, abneural HC in the middle part often had larger e¡erent contact areas in the studied species although typical cup-like synapses were rare. The number of a¡erent nerve terminals per HC is typically low in birds. In the emu, neural HC along the whole length of the BP all had a¡erent ¢bers, mostly one to three. In birds, most HC a¡erents are exclusive, i.e. one ¢ber innervates only one HC. In the BP of the emu this was only true for the basal 30% of the BP. Especially in its apex, many HC had non-exclusive a¡erent innervation, i.e. one a¡erent ¢ber contacted several neighboring HC. Koëppl and Manley (1997) obtained similar results by iontophoretically labeling primary a¡erents. In parallel with this high proportion of non-exclusive a¡erents, there are numerous contacts between neighboring HC. Thus, there appears to be a high degree of peripheral integration in the apex. These non-exclusive a¡erents and HC-HC contacts in a large proportion of the emu papilla seem to be a primitive feature. In the emu BP, this is restricted to a¡erents with best frequencies below 1 kHz (Fig. 9e). In other, and even in most specialized species like the barn owl, a comparable situation is found only in the most apical part of their papillae. The apex is considered to be the evolutionary oldest portion of the avian basilar papilla (Smith et al., 1985; Lavigne-Rebillard et al., 1985). In modern birds, like the song birds, the relative number of a¡erents has increased and thus the innervation density of the THC (for review see Gleich and Manley, 1998). This was much more the case in the high-frequency base of the avian papilla, and is especially elaborate in a high-frequency specialist, the barn owl, where the most neural HC may have up to 20 a¡erent terminals and thus reach the density of a¡erent innervation of mammalian IHC (Fischer, 1994a).
The majority of HC receive a¡erent innervation. The zone of the true SHC (HC without a¡erents, Fischer, 1994a,b) along the abneural edge of the papilla is rather narrow in the emu, and this is probably a primitive condition (Fig. 11). In species specialized for a higher frequency range, like the barn owl, the SHC are the dominating HC type in the BP's base. SHC obviously are important elements in higher-frequency hearing. However, a zone without a¡erent innervation is also present in the primitive emu, and this fact is corroborated by the failure to label primary a¡erents near the abneural edge of the emu BP (Koëppl and Manley, 1997). This points to a unique and common characteristic of the avian hearing epithelium. As far as we know, such avian abneural HC are the only sensory cells known to routinely lack a¡erent innervation. This o¡ered the possibility to clearly (and functionally) de¢ne SHC as those HC without a¡erent but with efferent innervation, and THC as those HC with a¡erent (and mostly also e¡erent) innervation (Fischer, 1994a). On the other hand, the `standard bird', the chicken, has, in its apical part of the BP, many abneural HC with a¡erent terminals. It is not clear whether this is rather a primitive state or an e¡ect of domestication. Di¡erent results, e.g. on the pattern of the size of e¡erent terminals (a gradient, or a rather sudden change in size) across the BP (Ofsie and Cotanche, 1996 ; Zidanic and Fuchs, 1996 ; Fischer, 1992), point to some variation between domesticated chickens. One should be cautious in considering the chicken BP as `the typical avian BP'. The THC are more similar to typical HC of more primitive vertebrate groups (Takasaka and Smith, 1971 ; Chandler, 1984). They perform the original sensory function, i.e. processing information and transmitting it towards the brain (Manley et al., 1989; Gleich, 1989 ; Manley and Gleich, 1992). A large proportion of the THC (and especially the most neural ones which have the largest a¡erent innervation) lie over the cartilage-like neural limbus, and not over the free (movable) basilar membrane. For this reason, the tectorial membrane must play a crucial role in the mechanical stimulation of the THC. Like the OHC in mammals, a derived hair cell type has evolved in birds (SHC); they always lie over the basilar membrane. As the function of the SHC cannot be sensory (in contrast to the mammalian OHC), they probably are e¡ectors that can be in£uenced by their e¡erents. They possibly change the mechanics of the tectorial membrane, e.g. by a change in sti¡ness or by bundle movements, and they also could modify the stimulus on the THC ; in this way, the SHC would play a functionally supportive role, as it is also proposed for the OHC in mammals (see e.g. Ashmore, 1994). The zone of hyaline cells along the abneural edge of the papilla is very broad in the emu. These hyaline cells
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were studied in detail in the chicken ; they contain actin and myosin in a regular array (Cotanche et al., 1992). They receive en passant innervation from the e¡erents that enter the basilar papilla through the habenula perforata (Oesterle et al., 1992). After contacting the hyaline cells, e¡erent ¢bers turn back and innervate the SHC (Zidanic and Fuchs, 1996). Therefore it is reasonable to assume that the SHC and the hyaline cells together form a functionally supporting system in the avian BP. In the emu, the hyaline cells are prominent while the SHC zone is rather small. Detailed analyses of the SHC/hyaline cell system are needed to obtain a better knowledge of this supposed supporting apparatus in the avian cochlea. Acknowledgments I am very grateful to O. Gleich and C. Koëppl for the ¢xed and embedded specimens. Many thanks also to C. Koëppl, H.-J. Leppelsack and G.A. Manley for commenting on an earlier version of this paper. I especially thank G.A. Manley for his support and the correction of my English. The photographic work of Gaby Schwabedissen is gratefully acknowledged. The Deutsche Forschungsgemeinschaft (DFG) supported this and other comparative studies with a grant to G.A. Manley within the SFB 204 (`Gehoër'). Many thanks are also due to three anonymous reviewers for their constructive comments.
References Ashmore, J.F., 1994. The cellular machinery of the cochlea. Exp. Physiol. 79, 113^134. Brix, J., Fischer, F.P., Manley, G.A., 1994. The cuticular plate of the hair cell in relation to morphological gradients of the chicken basilar papilla. Hear. Res. 75, 244^256. Carrol, R.L., 1988. Vertebrate Palaeontology and Evolution. Freeman, New York. Chandler, J.P., 1984. Light and electron microscopic studies of the basilar papilla in the duck, Anas platyrhynchos: I. The hatchling. J. Comp. Neurol. 222, 506^522. Chen, L., Salvi, R., Shero, M., 1994. Cochlear frequency-place map in adult chickens: Intracellular biocytin labeling. Hear. Res. 81, 130^ 136. Code, R.A., Carr, C.E., 1994. Choline acetyltransferase-immunoreactive cochlear e¡erent neurons in the chick auditory brainstem. J. Comp. Neurol. 340, 161^173. Cole, K.S., Gummer, A.W., 1990. A double-label study of e¡erent projections to the cochlea of the chicken, Gallus domesticus. Exp. Brain Res. 82, 585^588. Corwin, J.T., Cotanche, D.A., 1988. Regeneration of sensory hair cells after acoustic trauma. Science 240, 1772^1774. Cotanche, D.A., 1987. Regeneration of the tectorial membrane in the chick cochlea following severe acoustic trauma. Hear. Res. 30, 197^206. Cotanche, D.A., Henson, M.M., Henson, O.W., 1992. Contractile
123
proteins in the hyaline cells of the chicken cochlea. J. Comp. Neurol. 324, 353^364. Cracraft, J., 1981. Towards a phylogenetic classi¢cation of recent birds of the world (class Aves). Auk 98, 681^714. Dannhof, B.J., Roth, B., Bruns, V., 1991. Length of hair cells as a measure of frequency representation in the mammalian inner ear? Naturwissenschaften 78, 570^573. Feduccia, A., 1980. The Age of Birds. Harvard University Press, Cambridge, MA. Fischer, F.P., 1992. Quantitative analysis of the innervation of the chicken basilar papilla. Hear. Res. 61, 167^178. Fischer, F.P., 1994a. Quantitative TEM analysis of the barn owl basilar papilla. Hear. Res. 73, 1^15. Fischer, F.P., 1994b. General pattern and morphological specializations of the avian cochlea. Scan. Microsc. 8, 351^364. Fischer, F.P., 1994c. Morphology and innervation pattern of hair cells in the basilar papilla of the emu. Abstracts of the 31th Inner Ear Biology Workshop, Montpellier, 10-13 September 1994, p. 12. Fischer, F.P., Koëppl, C., Manley, G.A., 1988. The basilar papilla of the barn owl Tyto alba: A quantitative morphological SEM analysis. Hear. Res. 34, 87^101. Fischer, F.P., Brix, J., Singer, I., Miltz, C., 1991. Contacts between hair cells in the avian cochlea. Hear. Res. 53, 281^292. Fischer, F.P., Singer, I., Miltz, C., Manley, G.A., 1992. Morphological gradients in the starling basilar papilla. J. Morphol. 213, 225^ 240. Fuchs, P.A., 1992. Ionic currents in cochlear hair cells. Prog. Neurobiol. 39, 493^505. Fuchs, P.A., Murrow, B.W., 1992. Cholinergic inhibition of short (outer) hair cells of the chick's cochlea. J. Neurosci. 12 (3), 800^ 809. Gleich, O., 1989. Auditory primary a¡erents in the starling: Correlation of function and morphology. Hear. Res. 37, 255^268. Gleich, O., Manley, G.A., 1998. The Hearing Organ of Birds and their Relatives. Springer, Berlin (in press). Gleich, O., Manley, G.A., Mandl, A., Dooling, R., 1994. The basilar papilla of the canary and the zebra ¢nch: a quantitative scanning electron microscopic description. J. Morphol. 221, 1^24. Jones, S.M., Jones, T.A., 1995. The tonotopic map of the embryonic chick cochlea. Hear. Res. 82, 149^157. JÖrgensen, J.M., Christensen, J.T., 1989. The inner ear of ther common rhea (Rhea americana L.). Brain Behav. Evol. 34, 273^280. Kaiser, A., Manley, G.A., 1994. Physiology of putative single cochlear e¡erents in the chicken. J. Neurophysiol. 72, 2966^2979. Klinke, R., Smolders, J.W.T., 1993. Performance of the avian inner ear. In: Allum, J.H.J., Allum-Mecklenburg, D.J., Harris, F.P., Probst, R. (Eds.), Progress in Brain Research, Vol. 97, pp. 31^ 43. Koëppl, C., Manley, G.A., 1997. Frequency representation in the emu basilar papilla. J. Acoust. Soc. Am. 101, 1574^1584. Koëppl, C., Gleich, O., Manley, G.A., 1993. An auditory fovea in the barn owl cochlea. J. Comp. Physiol. 171, 695^704. Lavigne-Rebillard, M., Cousillas, H., Pujol, R., 1985. The very distal part of the basilar papilla in the chicken: A morphological approach. J. Comp. Neurol. 238, 340^347. Manley, G.A., 1995. The avian hearing organ: a status report. In: Manley, G.A., Klump, G.M., Koëppl, C., Fastl, H., Oeckinghaus, H. (Eds.), Advances in Hearing Research. World Scienti¢c, Singapore, pp. 219^229. Manley, G.A., Gleich, O., 1992. Evolution and specialization of function in the avian auditory periphery. In: Webster, D.B., Fay, R., Popper, A.N. (Eds.), The Evolutionary Biology of Hearing. Springer, Berlin. Manley, G.A., Brix, J., Kaiser, A., 1987. Developmental stability of the tonotopic organization of the chick's basilar papilla. Science 237, 655^656. Manley, G.A., Gleich, O., Kaiser, A., Brix, J., 1989. Functional di¡er-
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124
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entiation of sensory cells in the avian auditory periphery. J. Comp. Physiol. A 164, 289^296. Manley, G.A., Meyer, B., Fischer, F.P., Schwabedissen, G., Gleich, O., 1996. Surface morphology of the basilar papilla of the tufted duck Aythya fuligula and the domestic chicken Gallus gallus domesticus. J. Morphol. 227, 197^212. Manley, G.A., Koëppl, C., Yates, G.K., 1997. Activity of primary auditory neurones in the cochlear ganglion of the Emu Dromaius novaehollandiae I: Spontaneous discharge, frequency tuning and phase locking. J. Acoust. Soc. Am. 101, 1560^1573. Marchant, S., Higgins, P.J., 1990. Handbook of Australian, New Zealand and Antarctic Birds. Oxford University Press, Melbourne, pp. 47^58. Oesterle, E.C., Cunningham, D.E., Rubel, E.W., 1992. Ultrastructure of hyaline, border, and vacuole cells in chick inner ear. J. Comp. Neurol. 318, 64^82. Ofsie, M.S., Cotanche, D.A., 1996. Distribution of nerve ¢bers in the basilar papilla of normal and sound-damaged chick cochleae. J. Comp. Neurol. 370, 281^294.
Pujol, R., 1991. Is the length of OHCs correlated with the frequency coding of the cochlea? Midwinter Meeting, Assoc. Res. Otolaryngol., St. Petersburg, FL, p. 125. Ryals, B.M., Rubel, E.W., 1988. Hair cell regeneration after acoustic trauma in adult Coturnix quail. Science 240, 1774^1776. Smith, C.A., Konishi, M., Schull, N., 1985. Structure of the barn owl's (Tyto alba) inner ear. Hear. Res. 17, 237^247. Smolders, J.W.T., Ding-Pfennigdor¡, D., Klinke, R., 1995. A functional map of the pigeon basilar papilla: correlation of the properties of single auditory nerve ¢bres and their peripheral origin. Hear. Res. 92, 151^169. Takasaka, T., Smith, C.A., 1971. The structure and innervation of the pigeon's basilar papilla. J. Ultrastruct. Res. 35, 20^65. Tanaka, K., Smith, C.A., 1978. Structure of the chicken's inner ear: SEM and TEM study. Am. J. Anat. 153, 251^271. Zidanic, M., Fuchs, P.A., 1996. Synapsin-like immunoreactivity in the chick-cochlea: speci¢c labeling of e¡erent nerve terminals. Audit. Neurosci. 2, 347^362.
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