Combinatorial enzymatic digestion with thermolysin and collagenase type I improved the isolation and culture effects of hair cell progenitors from rat cochleae.

The high incidence of hearing loss in human combined with the lack of hair cell regeneration in mammalian cochleae had got the attention to manipulate stem/progenitor cells to participate in hair cell regeneration for years. Cochlear progenitor cells are considered as the best candidate for hair cell regeneration. However, there is not any effective and feasible way to separate hair cell progenitors from rat cochleae, yet. In this study, we tried to isolate single epithelial cells from rat basilar membrane by combinatorial enzymatic digestion with thermolysin and collagenase type I. The results showed that the harvested single cells gave rise to otospheres with features of stem cells and could be induced to differentiate into hair cells. Significantly, more otospheres of epithelial origin were obtained by digesting with thermolysin and collagenase type I. The combinatorial enzymatic digestion would be a potential method for hair cell progenitor isolation and culture with broad applications.

Effect of Growth Factor Supplementation on the Hair Cell Specific Markers of Cells Harvested from Basilar Membrane.

OBJECTIVE: Loss of auditory hair cells is a major cause of deafness. The presence of auditory progenitor cells in the inner ear raises the hope for mammalian inner ear cell regeneration. In this study, we aimed to investigate the effect of growth factor supplementations, namely a combination of epidermal growth factor (EGF), insulin-like growth factor (IGF), and beta (ß)-fibroblast growth factor (ßFGF), on the expression of hair cell-specific markers by cells harvested from the cochlear membrane. This would provide an insight into the capability of these cells to differentiate into hair cells. MATERIALS AND METHODS: EGF, IGF, and ßFGF were supplemented into the culture medium. The cells were evaluated by morphology, growth kinetic, gene expression, and protein expression. RESULTS: The cultured cells of mouse basilar membrane were spindle shaped. Growth factors-enriched medium promotes a significantly higher proliferative activity than the basic culture medium but did not alter the cell morphology. Growth factors-enriched medium did not show any significant differences in the protein expression of the hair cell-specific markers myosin VIIa and calretinin and the stem-cell marker nestin. Gene expression analysis showed that the expression of the hair cell-specific genes myosin VIIa and calretinin as well as the stem cell genes nestin, Rex1, and Sox2 was reduced after the cells were passaged in the growth factor-supplemented medium. Cells in the basic medium expressed a significantly higher level of hair cell-specific genes at certain passages. CONCLUSION: Growth factor supplementation could not maintain the expression of hair cell-specific markers by cells obtained from the cochlear membrane.

Activation of the antigen presentation function of mononuclear phagocyte populations associated with the basilar membrane of the cochlea after acoustic overstimulation.

The immune response is an important component of the cochlear response to stress. As an important player in the cochlear immune system, the basilar membrane immune cells reside on the surface of the scala tympani side of the basilar membrane. At present, the immune cell properties in this region and their responses to stress are not well understood. Here, we investigated the functional role of these immune cells in the immune response to acoustic overstimulation. This study reveals that tissue macrophages are present in the entire length of the basilar membrane under steady-state conditions. Notably, these cells in the apical and the basal sections of the basilar membrane display distinct morphologies and immune protein expression patterns. Following acoustic trauma, monocytes infiltrate into the region of the basilar membrane, and the infiltrated cells transform into macrophages. While monocyte infiltration and transformation occur in both the apical and the basal sections of the basilar membrane, only the basal monocytes and macrophages display a marked increase in the expression of major histocompatibility complex (MHC) II and class II transactivator (CIITA), a MHC II production cofactor, suggesting the site-dependent activation of antigen-presenting function. Consistent with the increased expression of the antigen-presenting proteins, CD4(+) T cells, the antigen-presenting partner, infiltrate into the region of the basilar membrane where antigen-presenting proteins are upregulated. Further pathological analyses revealed that the basal section of the cochlea displays a greater level of sensory cell damage, which is spatially correlated with the region of antigen-presenting activity. Together, these results suggest that the antigen-presenting function of the mononuclear phagocyte population is activated in response to acoustic trauma, which could bridge the innate immune response to adaptive immunity.

Coupling active hair bundle mechanics, fast adaptation, and somatic motility in a cochlear model.

One of the central questions in the biophysics of the mammalian cochlea is determining the contributions of the two active processes, prestin-based somatic motility and hair bundle (HB) motility, to cochlear amplification. HB force generation is linked to fast adaptation of the transduction current via a calcium-dependent process and somatic force generation is driven by the depolarization caused by the transduction current. In this article, we construct a global mechanical-electrical-acoustical mathematical model of the cochlea based on a three-dimensional fluid representation. The global cochlear model is coupled to linearizations of nonlinear somatic motility and HB activity as well as to the micromechanics of the passive structural and electrical elements of the cochlea. We find that the active HB force alone is not sufficient to power high frequency cochlear amplification. However, somatic motility can overcome resistor-capacitor filtering by the basolateral membrane and deliver sufficient mechanical energy for amplification at basal locations. The results suggest a new theory for high frequency active cochlear mechanics, in which fast adaptation controls the transduction channel sensitivity and thereby the magnitude of the energy delivered by somatic motility.

Localization of fatty acid binding proteins (FABPs) in the cochlea of mice.

Various fatty acids (FAs) are involved in many different functions in the organism as a source of energy, as essential ingredients of membranous lipids as well as intracellular signaling molecules. Intracellular fatty acid binding proteins (FABPs) comprise a family of soluble lipid binding proteins with low molecular masses and which can make long chain FAs soluble to allow intracellular translocation in the aqueous cytosol. To clarify the possible involvement of FAs and FABPs in hearing function, the present study investigated the localization of FABPs in the cochlea of adult mice using immunohistochemical procedures. Among various FABP species, H (heart-type)-FABP was localized in inner and outer pillar cells and outer phalangeal cells, while B (brain-type)-FABP was localized in border cells and cells of Hensen, and fibrocytes in the spiral limbus and spiral prominence. In the spiral ganglion, moderate to low H-FABP immunoreactivity was observed in almost all neurons, while B-FABP immunoreactivity was found in satellite cells. The discrete localization of the two FABPs in different non-receptor cells in the Organ of Corti suggests that the FABP species and/or their ligands, FAs, play important roles in the regulation of the hearing function.

Transgenic BDNF induces nerve fiber regrowth into the auditory epithelium in deaf cochleae.

Sensory organs typically use receptor cells and afferent neurons to transduce environmental signals and transmit them to the CNS. When sensory cells are lost, nerves often regress from the sensory area. Therapeutic and regenerative approaches would benefit from the presence of nerve fibers in the tissue. In the hearing system, retraction of afferent innervation may accompany the degeneration of auditory hair cells that is associated with permanent hearing loss. The only therapy currently available for cases with severe or complete loss of hair cells is the cochlear implant auditory prosthesis. To enhance the therapeutic benefits of a cochlear implant, it is necessary to attract nerve fibers back into the cochlear epithelium. Here we show that forced expression of the neurotrophin gene BDNF in epithelial or mesothelial cells that remain in the deaf ear induces robust regrowth of nerve fibers towards the cells that secrete the neurotrophin, and results in re-innervation of the sensory area. The process of neurotrophin-induced neuronal regeneration is accompanied by significant preservation of the spiral ganglion cells. The ability to regrow nerve fibers into the basilar membrane area and protect the auditory nerve will enhance performance of cochlear implants and augment future cell replacement therapies such as stem cell implantation or induced transdifferentiation. This model also provides a general experimental stage for drawing nerve fibers into a tissue devoid of neurons, and studying the interaction between the nerve fibers and the tissue.

[Comparison of different chemical methods for preparation of acellular nerve scaffold].

OBJECTIVE: Compare the effect of different chemical methods for preparation of acellular nerve scaffold and to provide an effective nerve scaffold for tissue engineering. METHODS: Fifteen male SD rats of 2 months old, weighing 200-250 g were selected; the bilateral sciatic nerves were harvested and divided into 3 groups according to preparation methods: group A (normal nerve), group B (Sondell method) and group C (optimal method by Triton X-200, SB-10 and SB-16). The morphology was compared by HE, immunohistochemistry and SEM after dispose; the degrees of decellularization, degrees of demyelination and integrity of the nerve fiber tube were assessed by scoring system. RESULTS: HE staining: In group A, the cross section of nerve was roundness, the cell nuclei was dark blue and the myelin sheath was reticular structure. In group B, the axon and cell nuclei disappeared and the structure of endoneurium was destroyed. In group C, the axon and cell nuclei disappeared and the endoneurium become animalistic round cavum. The immunohistochemistry staining of Laminin: In group A, the myelin sheath was surrounded by basement membrane with dark blue SC nuclei inside. In group B, the myelin sheath and SC nuclei disappeared and the structure of basement membrane destroyed. In group C, the myelin sheath and SC nuclei disappeared and basement membrane become animalistic round cavum. The immunohistochemistry staining of S-100: In group A, the myelin sheath and SC were brown. In groups B and C, there were no apparent stained myelin sheath. SEM: In group A, the myelin sheath and axon were clear. In group B, the axon and myelin sheath disappeared and basement membrane became animalistic. In group C, the basement membrane was more regular than that of group B. The degrees of acellularization and demyelination of groups B and C were superior to that of group A (P < 0.05), and the degrees of demyelination of group C were superior to that of group B (P < 0.05). The integrity of nerve fiber tube of group C was superior to that of group B (P < 0.05) and similar to that of group A (P > 0.05). The total score was the lowest in group C but the quality was the best. CONCLUSION: The effect of decellularization of optimal method was similar to that of traditional Sondell method, but the effect of demyelination and integrity of nerve fiber tube were better than that of traditional Sondell method. And this acellular nerve can be used as a new kind of nerve scaffold material.

[Construction of recombinant adenoviral vector to coexpress human neurotrophin3 and EGFP gene and its conduction efficiency to rat cochlea in vitro].

OBJECTIVE: To construct an adenoviral vector that codes for both human NT3 and EGFP, to confirm the transduction efficiency in rat cochlear cultures and to assess the protection of NT3 on SGNs survival. METHOD: PAdeasy-1 and pAdTrack CMV were used to constructed Ad/NT3 adenovirus and then to transfer postnatal day 3 rat cochlear cultures. The transduction efficiency was determined by microscope observation. The amounts of SGNs were counted to evaluated protection of Ad/NT3 on SGNs survival. RESULT: EGFP positive cells were observed in all cochlear turns. There was approximately 49% in outer sulcus cells and 27% in the interdental cells; less than 2% of the hair cells and SGN. The amounts of SGN of treated Ad/NT3 adenovirus are more than cochlea SGN only Ad/EGFP adenovirus after cultured for 15 days. CONCLUSION: Ad/NT3 adenovirus could transduce EGFP and NT3 in large number of supporting cells, but few hair cells or SGNs. The putative release of NT3 from these supporting cells could enhance cell survival and promote neurite outgrowth from SGNs.

Tuning the cochlea: wave-mediated positive feedback between cells.

Frequency analysis by the mammalian cochlea is traditionally thought to occur via a hydrodynamically coupled 'travelling wave' along the basilar membrane. A persistent difficulty with this picture is how sharp tuning can emerge. This paper proposes, and models, a supplementary or alternative mechanism: it supposes that the cochlea analyses sound by setting up standing waves between parallel rows of outer hair cells. In this scheme, multiple cells mutually interact through positive feedback of wave-borne energy. Analytical modelling and numerical evaluation presented here demonstrate that this can provide narrow-band frequency analysis. Graded cochlear tuning will then rely on the distance between rows becoming greater as distance from the base increases (as exhibited by the actual cochlea) and on the wave's phase velocity becoming slower. In effect, tuning is now a case of varying the feedback delay between the rows, and a prime candidate for a wave exhibiting suitably graded phase velocity-a short-wavelength 'squirting wave'-is identified and used in the modelling. In this way, resonance between rows could supply both amplification and high Q, characteristics underlying the 'cochlear amplifier'-the device whose action has long been evident to auditory science but whose anatomical basis and mode of operation are still obscure.

The cochleogram of the guinea pig.

The cochleogram is an important tool to relate properties of the cochlea (e.g. hair cell loss, damaged hair cells) to their position in the cochlear turns, to calculate the average hair cell density, and to measure the length of the whole cochlea. In this work different methods of plotting cochleograms are compared. We suggest that a sector-wise division of the cochlea for counting a cochleogram has advantages over line diagrams that provide a higher spatial resolution but might lead to misinterpretations of the degree of missing hair cells. The scanning electron microscopic analysis of 171 guinea pig cochleas revealed a mean basilar membrane length of 16.4 +/- 1.4 mm (mean +/- standard deviation) with sector lengths of 6.9, 4.2, 3.2, and 1.9 mm, thus adding relevant information to the morphology of the guinea pig cochlea.

Local mechanical properties of mouse outer hair cells: atomic force microscopic study.

OBJECTIVES: Outer hair cells (OHCs) are capable of altering their cell length in response to changes in membrane potential. Due to this electromotility, OHCs probably subject the basilar membrane to force, resulting in cochlear amplification. To understand the mechanism of such amplification, knowledge of the mechanical properties of OHCs is required since the force produced by OHC electromotility is thought to depend on such properties. Various studies have been conducted to investigate the mechanical properties of guinea pig OHCs. With regard to mice, however, although various kinds of transgenic and knockout mice possess great potential as research models, the mechanical properties of mouse OHCs have not as yet been reported since the cells and/or tissues in the mouse hearing organ are relatively small and vulnerable to external stimuli, rendering sample preparation difficult. In this study, therefore, to establish indicators of the mechanical properties of OHCs in mice, such properties were measured by atomic force microscopy (AFM). METHODS: CBA/JNCrj strain male mice aged 10-12 weeks (25-30 g) were used. Cochleae were dissected out from the animal and both the basilar membrane and the organ of Corti were simultaneously unwrapped from the modiolus with forceps. Dissected coiled tissue was then incubated with an enzymatic digestion medium for 15 min. After digestion, OHCs were isolated by gently triturating the coiled tissue. Local mechanical properties of the OHCs were then measured by an indentation test using an AFM. RESULTS: Young's modulus and stiffness of the OHC in the apical turn of the mouse cochlea were 2.1+/-0.5 kPa and 4.4+/-1.2 mN/m, respectively. CONCLUSIONS: Young's modulus of the OHC in the apical turn of the cochlea in mice was roughly the same as that in the apical turn of the cochlea in guinea pigs; however, the stiffness of the former was about two times greater than that of the latter because the cell length of the former was shorter than that of the latter.

Probable function of Boettcher cells based on results of morphological study: localization of nitric oxide synthase.

Boettcher cells lie on the basilar membrane beneath Claudius cells. The cells are considered supporting cells for the organ of Corti, and present only in the lower turn of the cochlea, which responds to high-frequency sound. Boettcher cells interdigitate with each other, and project microvilli into the intercellular space. Their structural specialization suggests that Boettcher cells may play a significant role in the function of the cochlea. Nitric oxide synthase (NOS) has previously been detected in substructures of the cochlea. In the cochlea, it is believed that nitric oxide plays an important role in neurotransmission, blood flow regulation, and induction of cytotoxicity under pathological conditions. Findings concerning detection of NOS on Boettcher cells are rare. We demonstrated here the localization of NOS on Boettcher cells of the rat by immunohistochemistry using polyclonal antibody to NOS. On observation with the light microscope using DAB staining, positive immunostaining to NOS was observed in Boettcher cells. In immunoelectron micrographs, NOS was detected abundantly in the cytoplasm of the interdigitations. This suggests that the interdigitations may play significant roles by using NOS. It follows from this that the nitric oxide (NO) on Boettcher cells may influences neighboring Boettcher cells. The ultrastructure of Boettcher cells suggests that they may be active cells, which perform both secretory and absorptive functions.

Organ of Corti kinematics.

The internal workings of the organ of Corti and their relation to basilar membrane motion are examined with the aid of a simple kinematic model. It is shown that, due to the lever system embodied in the organ of Corti, there is a significant transformer gain between basilar membrane and cilia displacements. While this transformation is nonlinear, linear response prevails in the narrow physiologically relevant operating range of the ciliary transducer. The model also simulates cilia deflection when the mechanical stimulus is the length change of outer hair cells.

Internal shearing within the hearing organ evoked by basilar membrane motion.

The vibration of the hearing organ that occurs during sound stimulation is based on mechanical interactions between different cellular structures inside the organ of Corti. The exact nature of these interactions is unclear and subject to debate. In this study, dynamic structural changes were produced by stepwise alterations of scala tympani pressure in an in vitro preparation of the guinea pig temporal bone. Confocal images were acquired at each level of pressure. In this way, the motion of several structures could be observed simultaneously with high resolution in a nearly intact system. Images were analyzed using a novel wavelet-based optical flow estimation algorithm. Under these conditions, the reticular lamina moved as a stiff plate with a center of rotation in the region of the inner hair cells. Despite being enclosed in several types of supporting cells, the inner hair cells, together with the adjacent inner pillar cells, moved in a manner signifying high compliance. The outer hair cells displayed radial motion indicative of cellular bending. Together, these results show that shearing motion occurs between several parts of the organ, and that structural relationships within the organ change dynamically during displacement of the basilar membrane.

[Beta-actin changes during hair cell regeneration in the chick basilar papilla].

OBJECTIVE: To investigate the changes of beta-actin and beta-actin mRNA expression during hair cell regeneration in the chick basilar papilla(BP) following gentamicin ototoxicity. METHODS: Thirty-six chicks were given muscular injection of gentamicin at dosage of 100 mg/kg.d for 10 consecutive days, and twelve additional chicks received injection of normal saline instead of gentamicin as controls. At 1, 3, 7, 14, 21 and 28 days after the treatment, six chicks in experimental group and two chicks in control group were sacrificed. Basilar papillas were prepared for immunohistochemistry and in situ hybridization study. beta-actin and beta-actin mRNA expression in the damaged region were histologically analyzed by computer image system. RESULTS: beta-actin immunoreactivity of BP changed significantly after treatment with gentamicin. From the first day to the fourteenth day after gentamincin, beta-actin immunoreactivity turned positive gradually. It approximated the normal response level at the fourteenth day after treatment of gentamincin. Then increasing rate of beta-actin immunoreactivity positive response level slowed from the fourteenth day after the treatment. At seventh day after treatment, beta-actin mRNA expression in BP increased to peak value, which preceded the peak expression of beta-actin in the regenerated hair cells. CONCLUSION: Nascent hair cells seem to mature 14 days after hair cell damage, which may play a role in auditory functional recovery.

Hair cell regeneration in the chick basilar papilla after exposure to wide-band noise: evidence for ganglion cell involvement.

It has been demonstrated that the auditory epithelium in the chick basilar papilla may regenerate after acoustic or ototoxic damage. Both types of damage may elicit the appearance of new cells that may develop in to the sensory cells. Factors inducing this process and the role of ganglion cells, the first neuron cells in the auditory pathway, are still unknown. The pattern of auditory damage and regeneration, after octave-band and pure-tone noise exposure, has been well established in research studies on chicks, but there are scarce data on wide-band noise effects. The aim of this study was to investigate the effect of wide-band noise, with different exposure levels applied, on the chick basilar papilla and supporting cells. Further, it was also aimed to determine whether the proliferation of ganglion cells, after wide-band noise exposure, occurs. The morphological changes were assessed with fluorescent, light, and transmission electron microscopy. Cell proliferation was studied based on immunoreactivity assays of proliferating cell nuclear antigen (PCNA). The exposure to wide-band noise at 120 dB SPL for 72 h produced stripe-like lesion of tall hair cells along the neural edge of the basilar papilla, mainly in the middle and, at the lesser extend, in its proximal part. There was no patch-like damage to the region of short hair cells, commonly observed after the exposure to the octave-band or pure-tone noise. The lesion extend depended on the level of exposure. The lower equivalent levels of noise (120 dB SPL for 40 h intermittent exposure) produced proportionally less damage. No morphological changes at light and fluorescent microscopy (apart from tectorial membrane exfoliation) were observed at 110 dB SPL in case of 20 h intermittent exposure. The elimination of dying hair cells took place either by pulling a damaged cell down to the basilar membrane or by extruding the cell to the subtectorial space. New hair cells reappeared at the sensory epithelium on the fifth day after the end of exposure. Cell proliferation started prior to hair cell loss. PCNA-like immunoreactivity was observed after the exposure at all levels in both the damaged and intact areas. PCNA appeared not only in the supporting cells, as indicated in previous studies, but also in the ganglion cells, suggesting ganglion cell involvement in the process of regeneration.

Lateral interactions account for the pattern of the hair cell array in the chick basilar papilla.

It has been suggested that lateral interactions set up the array of hair cells and supporting cells in the chick basilar papilla. The presence of a hair cell would inhibit adjacent cells from becoming hair cells, and promote the formation of supporting cells. Models of cell specification were tested, starting with a closely packed array of multipotent progenitor cells. Lateral interactions, in which emerging hair cells promoted a supporting cell phenotype in adjacent cells, and in which emerging supporting cells promoted a hair cell phenotype in adjacent cells, produced an array of cells similar to that observed experimentally in the distal and central parts of the basilar papilla. In these areas, the ratio of supporting cells to hair cells is very close to 2:1, each hair cell on average being surrounded by six supporting cells, and each supporting cell being surrounded by three hair cells and three supporting cells. Identical patterns of hair and supporting cells could be produced by models in which either of the lateral inhibitory factors was replaced by a diffusive factor, i.e. a factor which acts on all cells in the model irrespective of position. The agreement of the model with observed cell ratios supports the view that the fate of both hair cells and supporting cells in the chick basilar papilla is a product of cell interactions within the papilla. It is possible that one factor, that provides contact lateral inhibition and promotes the formation of supporting cells, is the Notch/Delta system. It is possible that the other factor is retinoic acid, a diffusive factor that promotes the formation of hair cells.

Ontogenetic expression of trk neurotrophin receptors in the chick auditory system.

Neurotrophins and their cognate receptors are critical to normal nervous system development. Trk receptors are high-affinity receptors for nerve-growth factor (trkA), brain-derived neurotrophic factor and neurotrophin-4/5 (trkB), and neurotrophin-3 (trkC). We examine the expression of these three neurotrophin tyrosine kinase receptors in the chick auditory system throughout most of development. Trks were localized in the auditory brainstem, the cochlear ganglion, and the basilar papilla of chicks from embryonic (E) day 5 to E21, by using antibodies and standard immunocytochemical methods. TrkB mRNA was localized in brainstem nuclei by in situ hybridization. TrkB and trkC are highly expressed in the embryonic auditory brainstem, and their patterns of expression are both spatially and temporally dynamic. During early brainstem development, trkB and trkC are localized in the neuronal cell bodies and in the surrounding neuropil of nucleus magnocellularis (NM) and nucleus laminaris (NL). During later development, trkC is expressed in the cell bodies of NM and NL, whereas trkB is expressed in the nerve calyces surrounding NM neurons and in the ventral, but not the dorsal, dendrites of NL. In the periphery, trkB and trkC are located in the cochlear ganglion neurons and in peripheral fibers innervating the basilar papilla and synapsing at the base of hair cells. The protracted expression of trks seen in our materials is consistent with the hypothesis that the neurotrophins/tyrosine kinase receptors play one or several roles in the development of auditory circuitry. In particular, the polarized expression of trkB in NL is coincident with refinement of NM terminal arborizations on NL.

Nonlinear mechanics of the organ of Corti caused by Deiters cells.

Though the organ of Corti (OC) has been an object of experimental and theoretical hearing research, open questions remain concerning the processing of acoustic signals by the cochlea where the OC is located. Today there is extensive knowledge about single parts of the organ but a lack of understanding as to how these elements act together. One of the reasons for this is the missing analysis of the mechanics of the OC in three dimensions. In order to fill this gap, we have analyzed a short section (0.06 mm) of the basilar membrane including the OC and evaluated its nonlinear finite element model numerically. The Deiters cells are idealized as thin elastic beams with a comparably low modulus of elasticity of actin. Therefore, they show nonlinear mechanical behavior generating additional frequency components with two-tone stimulation.

Fine structure of the basilar papilla of the emu: implications for the evolution of avian hair-cell types.

The morphology of the basilar papilla of the emu was investigated quantitatively with light and scanning electron microscopical techniques. The emu is a member of the Paleognathae, a group of flightless birds that represent the most primitive living avian species. The comparison of the emu papilla with that of other, more advanced birds provides insights into the evolution of the avian papilla. The morphology of the emu papilla is that of an unspecialised bird, but shows the full range of features previously shown to be typical for the avian basilar papilla. For example, the orientation of the hair cells' sensitive axes varied in characteristic fashion both along and across the papilla. Many of the quantitative details correlate well with the representation of predominantly low frequencies along the papilla. The most distinctive features were an unusually high density of hair cells and an unusual tallness of the hair-cell bodies. This suggests that the evolution of morphologically very short hair cells, which are a hallmark of avian papillae, is a recent development in evolution. The small degree of differentiation in hair-cell size contrasts with the observation that a significant number of hair cells in the emu lack afferent innervation. It is therefore suggested that the development of functionally different hair-cell types in birds preceded the differentiation into morphologically tall and short hair cells.