The evolution of the hearing organs of amniote vertebrates

A very interesting conclusion can be reached from our work and the work of other groups: The hearing epithelia of the different groups of amniote vertebrates evolved independently from each other over a very long period of time (Manley and Köppl, 1998; Manley 2002, Manley and Clack, 2004). There are clear indications that at the beginning of amniote evolution (from the stem reptiles 375 million years ago), the hearing organ of amniotes were small and unspecialized. Initially, there was only one type of sensory hair cell. Following the branching-off in the Palaeozoic of the three main evolutionary lines of amniotes (synapsid, or mammal-like reptiles and two lines of diapsid reptiles leading (a) to the archosaurs, the ancestors of birds and Crocodilia and (b) to the lepidosaurs, the ancestors of lizards and snakes), each of these groups shows major changes in auditory-epithelial structure. Some of the developments were similar between the various groups, e.g., the epithelium became longer in all groups, and all groups developed two populations of hair cells. There are, however, also obvious differences. In lizards, the two hair-cell populations are found in different frequency areas of the papilla (i.e. separated along the papilla's length), whereas in mammals and birds, the hair-cell differentiation took place across the papilla and thus within each frequency area (see, e.g., Manley et al., 1989; Manley, 2004a; Gleich et al., 2004). These two populations are also different in birds and mammals, indicating that this development occurred independently in the two groups.

During the long period of independent evolution of these groups of amniotes (in some cases more than 300 million years), an enormous variety of structural constellations came into being, a variety that is not seen in any other of the major sense organs of vertebrates. Thus nature has created a large field of experimentation that provides optimal material to ask and to answer questions concerning structure-function relationships in the inner ear.

What was the performance of the early amniote hearing organs?

Comparative studies suggest that the hearing organ of the stem reptiles resembled the organ we see today in turtles and in Sphenodon, the Tuatara of New Zealand. It would have been less than 1mm long and have had perhaps a few hundred hair cells. The response activity of hair cells and afferent nerve fibres of primitive amniote hearing organs most likely already obeyed three important functional principles. These can be observed in all other amniotes and can be regarded as plesiomorphic characteristics (Manley, 2000):

First principle: A high frequency selectivity, achieved by two basic mechanisms:

a) An electrical resonance in the cell membranes of the sensory cells. Certain ion channels, when present in specific numbers and having specific time-delay characteristics in each hair cell, cause the membrane potential to oscillate with a specific, preferred frequency ('electrical tuning') when stimulated by sound. Due to the fact that a cell membrane can only incorporate a finite number of channels, this mechanism is confined to low frequencies, at high body temperatures perhaps 5 kHz.

b) A micromechanical resonance, most probably a later development, in which the preferred frequencies of the hair cells are determined by the properties of the stiffness of the stereovillar bundles of the hair cells and the mass of this bundle and any extracellular gel mass (tectorial structure) lying over them. The presence of this mechanism manifests itself in prominent anatomical gradients in the hearing epithelium (for example in the hair-cell bundle height).

Second principle: The hair cells are arranged systematically according to their preferred frequency (for both electrical and micromechanical tuning) along an axis of the hearing epithelium - i.e., a so-called tonotopic organisation is present (e.g., Köppl und Manley, 1990; Köppl und Manley, 1997; Köppl et al., 1993).

Third principle: There is active amplification in the hair cells. An increasing body of evidence indicates that all amniote hearing organs are capable of not only receiving sound stimuli, but also of amplifying weak stimuli. One bi-product of the amplification process is that some of the energy produced escapes the organ and can be measured as sound in the external ear canal. Our work has contributed to the knowledge that not only mammals, as originally thought, but birds and lizards, too, show phenomena associated with an amplification process. Especially the so-called spontaneous emissions, that have been found in all tetrapods (and are thus most likely a primitive property of hair cells; Köppl, 1995), are accepted as signs of the presence of an amplifier (Manley, 2001). The function of the amplifier is, via active movements of some part of the hair cell, to overcome the frictional viscosity of the watery medium in which the hair cells are found and thus increase hair-cell sensitivity. The behaviour of such otoacoustic emissions in the presence of external tonal stimuli can provide very interesting and detailed information on the function of the hair cells (see our contributions to the “Auditory Worlds…” book). Our studies are designed to help understand the significance of particular - often group-specific - morphological constellations on the function of the hearing organ.

What changes occurred in the hearing epithelium during evolution?

What fundamental changes in the structure of the hearing organs of the three main amniote lines, mammals, birds (and Crocodilia) and lizards (Manley, 2002) do we observe during the course of evolution (Köppl und Manley, 1991: Manley und Gleich, 1991; Manley, 2000, 2002)? There were both parallel developments, but also large differences. In fact, the hearing organs of the various representatives of these groups can each be immediately recognized as such from their morphology, which means that each group has a unique set of morphological features. There were, however, parallel developments, especially between birds and mammals. Two main developments were seen in all three groups:

1)    Electrical tuning became less important over time, and the frequency responses of the majority, and in some cases, all hair cells are now dominated by micromechanical tuning mechanisms. This mechanism has the advantage of not being restricted to frequencies below 5kHz, and this development was accompanied by an extension of the hearing range, especially in birds and mammals.

2)    The second development was an elongation of the papilla. This prominent elongation only makes sense in the context of micromechanical tuning, because hair cells tuned micromechanically need space to separate them if their movements are not going to interfere with each other. Whereas the primitive hearing epithelium was short, some lizards have epithelia that are 2mm long, some owls have epithelia that are more than 10mm long, and some mammals have epithelia of more than 50mm length. This elongation of the epithelium brought two advantages:

(A) An extension of the tonotopic frequency range without reducing the space available for frequencies already present. The space used per octave rose from about 0.2 mm in turtles, to over 0.5mm in some lizards, up to 5mm in birds (Köppl et al., 1993), and up to maximally 55 mm in some frequency ranges in specialized mammals. As smaller animals can produce, process and localize high frequencies better than low frequencies, this development was of great advantage.

(B) The second advantage is found in the fact that after elongation, there are more hair cells per frequency range, i.e., each frequency range can be analysed in more detail and the information passed to the brain in afferent fibres in a parallel-processing fashion. Mammals, for example, devote on average 800 hair cells to each octave. In some regions of particular behavioural interest, the innervation density is raised, increasing parallel processing even further.

These two developments are responsible for the fact that most modern amniotes possess highly-developed and selective hearing organs. These were, however, not the only evolutionary changes seen. In addition, there was a tendency to specialize hair cells into groups with different structure and function, and this tendency differed in its manifestation between amniote groups. In general, one can distinguish between two groups of hair cells, but these differ between the three amniote groups and developed independently in each group. The selection pressures on large hair-cell epithelia have in each case led to the evolution of larger hearing organs with specialised hair-cell populations (Manley, 2000). The functional meaning of the various hair-cell specialisations will in the future, provide rich material for comparative studies.

Readers interested in the evolution of hearing systems are referred to our chapters (see reference list) in the new book in the Springer Handbook of Auditory Research series Nr. 22:

Evolution of the Vertebrate Auditory System, (2004) Manley, G.A., Popper, A. and Fay, R.R. (eds) New York, Springer-Verlag.(Hardback: ISBN 0-387-21089-X: Paperback: ISBN 0-387-21093-8.

The evolution of the hearing organ of birds

Among the Archosauria – Birds (Aves) and Crocodilia, we also see an evolutionary elongation of the hearing epithelium and a specialisation of hair cells (Manley, 2000, 2002; Gleich et al., 2004). In this case, as in mammals, but not as in lizards, the specialisation is across the hearing organ, i.e., seen in all frequency ranges. Hair cells that lie on the inner, neural side differ in their shape, innervation and function from those on the outer, or abneural, side. The similarity to mammals, which are likely to be traceable to similar functional specialisations, indicate that each group reached a similar solution to the same evolutionary problem, that is, these are analog developments. One of the important differences, the innervational pattern, is even more distinct in birds than it is in mammals. Franz-Peter Fischer of our group discovered that in birds, many of the hair cells on the outer side of the epithelium (Short hair cells, or SHC) have no afferent nerve contacts whatsoever. This is a unique situation in sensory physiology. What is the function of sensory cells that have no way to send information to the brain?

In order to understand one possible function for the SHC, we need to be reminded of the ability of hair cells to produce mechanical energy. We strongly suspect that the hair-cell populations of both birds and mammals are the result of a specialisation for sensory reception on the one hand (inner hair cells of mammals, tall hair cells or THC of birds) and for amplification on the other hand (outer hair cells of mammals, SHC of birds). According to this notion, SHC would feed mechanical energy into the tectorial membrane, that would then transmit it to the THC. The THC in turn would tranduce the resulting net stimulus and transform it at the afferent synapse into a coded signal for the brain (Manley, 1995).

We were able to show that, in spite of some prominent structural differences, the inner ear of birds has a similar performance to that of mammals. There is, however, still a need to explore the detailed anatomy of the avian cochlea, which is still not as well understood as that of mammals. Especially important with regard to micromechanical tuning are the anatomical gradients along the Papilla basilaris, e.g., in the structure of the sensory cells and their innervation by afferent (to the brain) and efferent (arriving from the brain) nerve fibres. We intend to continue to use electron-microscopical serial sections to establish "maps" of the papillae of unspecialised and specialised bird species. Through a continued comparison of such data, we expect to be able to better understand the indicators for specific functions of the inner ear (Fischer, 1994; Köppl et al., 2000).

Our previous electrophysiological work on starlings, chickens (Manley et al., 1991), Emus (Manley et al., 1997) und barn owls (Köppl, 1997) showed that the response characteristics of auditory-nerve fibres of birds are very similar to those of mammals. This is true in spite of obvious morphological differences between these two types of hearing organ. This somewhat unexpected result makes it obvious that we do not really understand which functional mechanisms are the foundation of these responses. By further studying the ear of birds, we will attempt to examine in what way structure and function are correlated (Gleich und Manley, 1998; Gleich et al., 2004).

In cooperation with a research group in Western Australia (Dr. G.K. Yates), we carried out a detailed study of the anatomy and physiology of the hearing organ of a primitive bird, the emu (Manley et al., 1997). These data are important for understanding the evolution of the hearing organ of birds. The auditory responses in the emu differed from those of other birds, in that the frequency map is strictly logarithmic (Köppl and Manley, 1997), and single-fibre frequency-tuning curves were strongly asymmetrical, showing a mammal-like 'tail' on the low-frequency side. Both of these are probably primitive features. A study of the structure of hair cells in the emu gave important clues to the evolution of the different hair-cell types (Köppl et al., 1998).

An additional, parallel specialisation of birds and mammals is found in the efferent innervation pattern. In birds as in mammals, the heaviest efferent innervation is to the abneural hair cells, in this case to SHC. Because of this similarity, birds are also suitable for studies of the efferent system. We have previously shown that the somata of chicken efferents are to be found in four areas of the brain stem (Kaiser and Manley, 1994). We were also able to show that there are at least two groups of efferent fibres that can be distinguished by their response patterns to sound. In addition to one type that responds in the same way as the efferents of mammals, we found a second population of efferent fibres that were suppressed by sound.

The barn owl ear

Among the birds that have been studied in our group, the barn owl is a special case. This species is highly specialised for nocturnal hunting, and can, for example, localise and catch mice using only its hearing system. This unusual capability is the result of a number of specialisations in the auditory system, from the asymmetrical external ears, the inner ear through to the various brain centres. Anatomical studies, including our own, have demonstrated that the hearing epithelium of the barn owl is obviously different to that of typical birds. Although a lot is known about the processing of sound stimuli by the owl's brain (visit the lab of Mark Konishi), much less is known about the function of the inner ear. We carried out both anatomical and electrophysiological studies (details on research projects concerning the neurophysiology, anatomy and development of the auditory nerve of the barn owl and on response properties of auditory neurones in the cochlear nuclei can be found in the web pages of Dr. Christine Köppl). The owls we use are from our own breeding colony.

In the barn owl, we were also able to show that both spontaneous (Taschenberger and Manley, 1997) and distortion-product otoacoustic emissions occur (Taschenberger und Manley, 1998). As in mammals, contralateral sound stimulation could produce a reduction in the amplitude of otoacoustic emissions. However, increases in the amplitude - facilitation - occurred equally often. The degree of change in amplitude changed during the experiments, but could reach values of ±10 dB. The results of experiments in which contralateral pure tones were applied indicate that these effects are highly frequency selective.

The evolution of the hearing organs of lizards

Lizards offer an especially interesting approach to research on the inner ear, since the different families of lizards differ greatly - and in a family-specific way - with respect to the structure and development of the hearing organ (Manley und Köppl, 1992; Manley and Köppl, 1998; Manley 2004). The two hair-cell types in lizards are found in two different areas arranged at different positions along the epithelium. The basilar papilla of lizards is thus always divided into at least two hair-cell areas. One area contains hair cells that respond only to low frequencies (below about 1kHz) and which are probably predominantly electrically tuned. This area resembles the entire papilla in turtles and varies only little between lizard families - it is evolutionarily conservative. The other hair-cell area is micromechanically tuned and respond to frequencies above 1kHz. These hair cells are separated from the low-frequency area and there are one or two areas either apical or basal of the low-frequency area, or both. The greatest variability in the morphology of the lizard papilla lies in this phylogenetically new, high-frequency area (Manley, 2002).

Through a detailed comparison of the anatomy and performance of the hearing organ in different lizard families, we hope to gain an understanding of the influence of the structural variety on the function. These studies will include detailed anatomy, electrophysiological experiments, modelling of frequency responses based on the known structure and measurement of otoacoustic emissions in the ear canal (Köppl und Manley, 1993, 1994; Manley, 1997; Manley und Köppl, 1994; Manley und Gallo, 1997; Manley et al., 1996; van Dijk et al., 1996).

One of the aims of these studies is to better understand the mechanisms responsible for generating otoacoustic emissions in all vertebrates. Using lizard species with differing inner-ear structure, we were already able to describe a correlation between this structural variety and the patterns in the otoacoustic emissions. The most important question studied in recent work on hair cells concerns the nature of the molecular motors that produce the sound energy of the emissions. We were for the first time able to study this phenomenon in an in-vivo preparation and show that the molecular motors driving the active process in lizards are to be found in the stereovillar bundles of the hair cells (Manley et al., 2001).

For this, we used the unique configuration of the hair-cell bundles in lizards, where in the high-frequency region, the hair-cell bundles are oriented oppositely to each other. We used a very low-frequency, relatively loud sound to alternately bias the position of the bundles of the different hair-cell populations during alternating phases of the sound stimulus. During such stimuli, we electrically stimulated the hair cells at frequencies that were precise multiples of the sound frequency and measured the sound emissions they produced using a sensitive microphone in the ear canal. We were able to show by this means that, as expected and when electrically stimulated, the two oppositely-oriented hair-cell groups produce out-of-phase sound emissions when unbiased. These emissions almost perfectly cancel, so that in the unbiased state, only extremely small emissions can be measured. As soon as some sound biasing is present, the sound emissions become larger, as the balance between the sounds emitted from the two populations changes. When loud sounds are used, the biasing is so strong in appropriate phases of the sound stimulus that the emissions from the two hair-cell groups can be seen during alternate phases of the biasing signal. These emissions are of opposite phase – and this can only mean that the sounds produced by the (in this case electrically-driven) active process originate from activity of the hair-cell bundles and not the hair-cell membrane (Manley et al., 2001).

In mammals, it is thought that hair-cell motility is the result of changes in the molecular configuration of protein complexes that are present in tightly-packed concentrations in the lateral cell membrane of outer hair cells. Since whole-cell motility of this kind has only been demonstrated in outer hair cells and not in inner hair cells of mammals or in any non-mammalian auditory hair cell, we decided to examine the question as to whether non-mammalian hair cells have such tightly-packed membrane particles. We (myself and Prof. C. Köppl) collaborated with Prof. Andrew Forge of the Institute of Laryngology and Otology of University College, London, England, who has facilities for studying freeze-fractured membrane surfaces. We chose to study two species that are known to produce spontaneous otoacoustic emissions, the barn owl Tyto alba (see Taschenberger and Manley, 1997) and the tokay gecko Gekko gecko (see Manley et al., 1996). Our study explored the possibility of membrane-based motility in hair cells of non-mammals, by determining their density of intra-membrane particles. Replicas of freeze-fractured membrane were prepared from auditory hair cells of the two species and, for quantitative comparison, mammalian inner and outer hair cells, as well as vestibular hair cells were evaluated. Lizard and bird hair cells displayed median densities of 2360 and 1880 intra-membrane particles per square micrometer. This was not significantly different from the densities in vestibular and mammalian inner hair cells. However, it was only about half the density measured in mammalian outer hair cells. This suggests that non-mammalian hair cells do not possess high densities of motor protein in their membranes and are thus unlikely to be capable of so-called “somatic motility”. It thus appears as if the active mechanism in the hair-cell bundle, as described above, is the plesiomorphic condition but nonetheless highly effective in non-mammals.

More recently, we have continued studying active mechanism in the hearing organ of lizards. We have described the effect of ac and dc electrical currents on the spontaneous otoacoustic emissions (SOAE) (Manley and Kirk, 2002). The SOAE are not only sensitive to current in Scala media, their frequencies and amplitudes are affected by changes in calcium concentrations in the endolymph (Manley et al., 2004). This latter effect was predictable through considerations as to the mechanism driving the active process in the hair-cell bundle (see e.g. Hudspeth, 1997; Choe et al., 2001; Manley, 2001). Injections of BAPTA, a calcium chelating agent, into Scala media causes a drop in calcium levels and this change forces the frequencies of SOAE down. Changing the calcium concentration directly by infusing different fluids into the Scala media induced downward shifts of SOAE frequency for calcium concentrations lower than 1.2mM and upward frequency shifts for higher concentrations (Manley et al., 2004). Our studies (myself, Dr. Ulrike Sienkecht and Prof. Christine Köppl) imply not only that the generating mechanism is in some way sensitive to calcium levels, but also that the normal calcium concentration in the lizard endolymph is near 1 mM and thus much higher than in mammals. This is presumably due to the presence of the otoliths of the lagena macula within the cochlear duct, since the otoliths are only stable if sufficient calcium is in the surrounding fluid.

These studies of otoacoustic emissions in lizards were partly carried out using the Australian Bobtail skink and in cooperation with a research group in Australia (led by the late Dr. Graeme Yates and supported by the late Dr. Des Kirk) and with Dr. P. van Dijk in the Netherlands. Electrophysiological studies on Gekko, in which we were able to show that, as predicted, the tonotopic map of the inner ear of geckos is reversed compared to the normal state in amniotes, were carried out in cooperation with Prof. M. Sneary, San Jose State University, California (see Manley et al., 1999). Research visits to the Australian group were supported by grants from the Raine Medical Research Foundation at the University of Western Australia, by the Adam Haker Fonds and the Hans-Neuffer-Stiftung.

The evolution of the hearing organs of mammals

Of course, most hearing-research scientists, if they are at all interested in the evolution of hearing in vertebrates, have the greatest interest in mammals. In this regard, it was clear very early on, that the ancestral mammalian hearing epithelium must have strongly resembled other ancestral hearing organs, since they have a common origin. The typical differences between the hearing organs of modern representatives of the various lineages are thus the result of many millions of years of separate evolution (Manley, 1971, 1973, 1986, 2000c; Manley and Köppl, 1998; Manley and Clack, 2004). In the case of mammals, there are substantial differences between hearing-organ structures in the different modern lineages (egg-laying monotremes versus therians - pouched - marsupial - and placental mammals; Manley, 2012c). Thus these differences have arisen in more recent times, beginning with a very early split in the mammalian lineages. The fact that all modern mammals show clear inner and outer hair cells separated by pillar cells indicates that this was a characteristic of the basal mammalian lineage. Monotreme mammals retained more of the ancestral condition, in which the hearing organ lies free in a more-or-less linear bony tube; these mammals, as most non-mammals - do not hear high frequencies. In the other groups- making up the therian mammals - a fundamental difference arose that proved to be extremely important in the later evolution of high-frequency hearing. That difference was the integration of the soft-tissue structures of the hearing organ with the framework of the surrounding bone. This added stiffness to the organ and made possible the evolution of responses to higher - and ultimately untrasonic (>20 kHz) frequency - hearing (Manley, 2012b). During the same geological periods, the therian lineages also evolved a specialized, three-ossicle middle-ear system that was pre-adapted to respond to very high frequencies (Manley, 2010a). Thus inner and middle ears evolved congruently, enabling an expansion to the audibility of very high frequencies in most - especially small - mammals. In some lineages, especially (but not exclusively) those with large body size, high-frequency hearing was lost in favour of greater frequency resolution at low frequencies. This happened in the primate lineage, resulting in the fact that humans have a low upper-frequency limit (see in this regard the controversy in Manley, 2010a, 2010c,2012b) and Heffner, H.E., Heffner, R.S., 2010. Response to Manley: an evolutionary perspective on middle ears. Hear Res 270, 1).

The evolution of the various types of middle ears and cochleae of the different lineages of modern mammals is thus not the result of a straight-forward, uniform path. The ideas behind the evolution of these structures thus need careful and differentiated study (Manley, 2012b).