The role of jab1, a putative downstream effector of the neurotrophic cytokine macrophage migration inhibitory factor (MIF) in zebrafish inner ear hair cell development.

Macrophage migration inhibitory factor (MIF) is a neurotrophic cytokine essential for inner ear hair cell (HC) development and statoacoustic ganglion (SAG) neurite outgrowth, and SAG survival in mouse, chick and zebrafish. Another neurotrophic cytokine, Monocyte chemoattractant protein 1 (MCP1) is known to synergize with MIF; but MCP1 alone is insufficient to support mouse/chick SAG neurite outgrowth or neuronal survival. Because of the relatively short time over which the zebrafish inner ear develops (~30hpf), the living zebrafish embryo is an ideal system to examine mif and mcp1 cytokine pathways and interactions. We used a novel technique: direct delivery of antisense oligonucleotide morpholinos (MOs) into the embryonic zebrafish otocyst to discover downstream effectors of mif as well as to clarify the relationship between mif and mcp1 in inner ear development. MOs for mif, mcp1 and the presumptive mif and mcp1 effector, c-Jun activation domain-binding protein-1 (jab1), were injected and then electroporated into the zebrafish otocyst 25-48hours post fertilization (hpf). We found that although mif is important at early stages (before 30hpf) for auditory macular HC development, jab1 is more critical for vestibular macular HC development before 30hpf. After 30hpf, mcp1 becomes important for HC development in both maculae.

Elastic force restricts growth of the murine utricle.

Dysfunctions of hearing and balance are often irreversible in mammals owing to the inability of cells in the inner ear to proliferate and replace lost sensory receptors. To determine the molecular basis of this deficiency we have investigated the dynamics of growth and cellular proliferation in a murine vestibular organ, the utricle. Based on this analysis, we have created a theoretical model that captures the key features of the organ's morphogenesis. Our experimental data and model demonstrate that an elastic force opposes growth of the utricular sensory epithelium during development, confines cellular proliferation to the organ's periphery, and eventually arrests its growth. We find that an increase in cellular density and the subsequent degradation of the transcriptional cofactor Yap underlie this process. A reduction in mechanical constraints results in accumulation and nuclear translocation of Yap, which triggers proliferation and restores the utricle's growth; interfering with Yap's activity reverses this effect.

Ontogenetic development of the inner ear saccule and utricle in the Lusitanian toadfish: Potential implications for auditory sensitivity.

Studies addressing structure-function relationships of the fish auditory system during development are sparse compared to other taxa. The Batrachoididae has become an important group to investigate mechanisms of auditory plasticity and evolution of auditory-vocal systems. A recent study reported ontogenetic improvements in the inner ear saccule sensitivity of the Lusitanian toadfish, Halobatrachus didactylus, but whether this results from changes in the sensory morphology remains unknown. We investigated how the macula and organization of auditory receptors in the saccule and utricle change during growth in this species. Inner ear sensory epithelia were removed from the end organs of previously PFA-fixed specimens, from non-vocal posthatch fry (<1.4 cm, standard length) to adults (>23 cm). Epithelia were phalloidin-stained and analysed for area, shape, number and orientation patterns of hair cells (HC), and number and size of saccular supporting cells (SC). Saccular macula area expanded 41x in total, and significantly more (relative to body length) among vocal juveniles (2.3-2.9 cm). Saccular HC number increased 25x but HC density decreased, suggesting that HC addition is slower relative to epithelial growth. While SC density decreased, SC apical area increased, contributing to the epithelial expansion. The utricule revealed increased HC density (striolar region) and less epithelial expansion (5x) with growth, contrasting with the saccule that may have a different developmental pattern due to its larger size and main auditory functions. Both macula shape and HC orientation patterns were already established in the posthatch fry and retained throughout growth in both end organs. We suggest that previously reported ontogenetic improvements in saccular sensitivity might be associated with changes in HC number (not density), size and/or molecular mechanisms controlling HC sensitivity. This is one of the first studies investigating the ontogenetic development of the saccule and utricle in a vocal fish and how it potentially relates to auditory enhancement for acoustic communication.

ADAM10 and γ-secretase regulate sensory regeneration in the avian vestibular organs.

The loss of sensory hair cells from the inner ear is a leading cause of hearing and balance disorders. The mammalian ear has a very limited ability to replace lost hair cells, but the inner ears of non-mammalian vertebrates can spontaneously regenerate hair cells after injury. Prior studies have shown that replacement hair cells are derived from epithelial supporting cells and that the differentiation of new hair cells is regulated by the Notch signaling pathway. The present study examined molecular influences on regeneration in the avian utricle, which has a particularly robust regenerative ability. Chicken utricles were placed in organotypic culture and hair cells were lesioned by application of the ototoxic antibiotic streptomycin. Cultures were then allowed to regenerate in vitro for seven days. Some specimens were treated with small molecule inhibitors of γ-secretase or ADAM10, proteases which are essential for transmission of Notch signaling. As expected, treatment with both inhibitors led to increased numbers of replacement hair cells. However, we also found that inhibition of both proteases resulted in increased regenerative proliferation. Subsequent experiments showed that inhibition of γ-secretase or ADAM10 could also trigger proliferation in undamaged utricles. To better understand these phenomena, we used RNA-Seq profiling to characterize changes in gene expression following γ-secretase inhibition. We observed expression patterns that were consistent with Notch pathway inhibition, but we also found that the utricular sensory epithelium contains numerous γ-secretase substrates that might regulate cell cycle entry and possibly supporting cell-to-hair cell conversion. Together, our data suggest multiple roles for γ-secretase and ADAM10 in vestibular hair cell regeneration.

Co-regulation of the Notch and Wnt signaling pathways promotes supporting cell proliferation and hair cell regeneration in mouse utricles.

This work sought to determine the crosstalk between the Notch and Wnt signaling pathways in regulating supporting cell (SC) proliferation and hair cell (HC) regeneration in mouse utricles. We cultured postnatal day (P)3 and P60 mouse utricles, damaged the HCs with gentamicin, and treated the utricles with the γ-secretase inhibitor DAPT to inhibit the Notch pathway and with the Wnt agonist QS11 to active the Wnt pathway. We also used Sox2-CreER, Notch1-flox (exon 1), and Catnb-flox (exon 3) transgenic mice to knock out the Notch pathway and activate the Wnt pathway in Sox2+ SCs. Notch inhibition alone increased SC proliferation and HC number in both undamaged and damaged utricles. Wnt activation alone promoted SC proliferation, but the HC number was not significantly increased. Here we demonstrated the cumulative effects of Notch inhibition and Wnt activation in regulating SC proliferation and HC regeneration. Simultaneously inhibiting Notch and overexpressing Wnt led to significantly greater SC proliferation and greater numbers of HCs than manipulating either pathway alone. Similar results were observed in the transgenic mice. This study suggests that the combination of Notch inhibition and Wnt activation can significantly promote SC proliferation and increase the number of regenerated HCs in mouse utricle.

Temporal coupling between specifications of neuronal and macular fates of the inner ear.

The inner ear is a complex organ comprised of various specialized sensory organs for detecting sound and head movements. The timing of specification for these sensory organs, however, is not clear. Previous fate mapping results of the inner ear indicate that vestibular and auditory ganglia and two of the vestibular sensory organs, the utricular macula (UM) and saccular macula (SM), are lineage related. Based on the medial-lateral relationship where respective auditory and vestibular neuroblasts exit from the otic epithelium and the subsequent formation of the medial SM and lateral UM in these regions, we hypothesized that specification of the two lateral structures, the vestibular ganglion and the UM are coupled and likewise for the two medial structures, the auditory ganglion and the SM. We tested this hypothesis by surgically inverting the primary axes of the otic cup in ovo and investigating the fate of the vestibular neurogenic region, which had been spotted with a lipophilic dye. Our results showed that the laterally-positioned, dye-associated, vestibular ganglion and UM were largely normal in transplanted ears, whereas both auditory ganglion and SM showed abnormalities suggesting the lateral but not the medial-derived structures were mostly specified at the time of transplantation. Both of these results are consistent with a temporal coupling between neuronal and macular fate specifications.

Hearing Assessment in Zebrafish During the First Week Postfertilization.

The zebrafish (Danio rerio) is a valuable vertebrate model for human hearing disorders because of many advantages in genetics, embryology, and in vivo visualization. In this study, we investigated auditory function of zebrafish during the first week postfertilization using microphonic potential recording. Extracellular microphonic potentials were recorded from hair cells in the inner ear of wild-type AB and transgenic Et(krt4:GFP)(sqet4) zebrafish at 3, 5, and 7 days postfertilization in response to 20, 50, 100, 200, 300, and 400-Hz acoustic stimulation. We found that microphonic threshold significantly decreased with age in zebrafish. However, there was no significant difference of microphonic responses between wild-type and transgenic zebrafish, indicating that the transgenic zebrafish have normal hearing like wild-type zebrafish. In addition, we observed that microphonic threshold did not change with the recording electrode location. Furthermore, microphonic threshold increased significantly at all tested stimulus frequencies after displacement of the saccular otolith but only increased at low frequencies after displacement of the utricular otolith, showing that the saccule rather than the utricle plays the major role in larval zebrafish hearing. These results enhance our knowledge of early development of auditory function in zebrafish and the factors affecting hearing assessment with microphonic potential recording.

Spatial and Age-Dependent Hair Cell Generation in the Postnatal Mammalian Utricle.

Loss of vestibular hair cells is a common cause of balance disorders. Current treatment options for bilateral vestibular dysfunction are limited. During development, atonal homolog 1 (Atoh1) is sufficient and necessary for the formation of hair cells and provides a promising gene target to induce hair cell generation in the mammals. In this study, we used a transgenic mouse line to test the age and cell type specificity of hair cell induction in the postnatal utricle in mice. We found that forced Atoh1 expression in vivo can induce hair cell formation in the utricle from postnatal days 1 to 21, while the efficacy of hair cell induction is progressively reduced as the animals become older. In the utricle, the induction of hair cells occurs both within the sensory region and in cells in the transitional epithelium next to the sensory region. Within the sensory epithelium, the central region, known as the striola, is most subjective to the induction of hair cell formation. Furthermore, forced Atoh1 expression can promote proliferation in an age-dependent manner that mirrors the progressively reduced efficacy of hair cell induction in the postnatal utricle. These results suggest that targeting both cell proliferation and Atoh1 in the utricle striolar region may be explored to induce hair cell regeneration in mammals. The study also demonstrates the usefulness of the animal model that provides an in vivo Atoh1 induction model for vestibular regeneration studies.

Ontogenetic development of the auditory sensory organ in zebrafish (Danio rerio): changes in hearing sensitivity and related morphology.

Zebrafish (Danio rerio) is an important model organism in hearing research. However, data on the hearing sensitivity of zebrafish vary across different reports. In the present study, the hearing sensitivity of zebrafish was examined by analysing the auditory evoked potentials (AEPs) over a range of total lengths (TLs) from 12 to 46 mm. Morphological changes in the hair cells (HCs) of the saccule (the main auditory end organ) and their synapses with primary auditory neurons were investigated. The AEPs were detected up to a much higher frequency limit (12 kHz) than previously reported. No significant difference in the frequency response range was observed across the TL range examined. However, the AEP thresholds demonstrated both developmental improvement and age-related loss of hearing sensitivity. The changes in hearing sensitivity were roughly consistent with the morphological changes in the saccule including (1) the number and density of HCs, (2) the organization of stereocilia, and (3) the quantity of a main ribbon protein, Ribeye b. The results of this study established a clear baseline for the hearing ability of zebrafish and revealed that the changes in the saccule contribute to the observed changes in TL (age)-related hearing sensitivity.

Gene Expression by Mouse Inner Ear Hair Cells during Development.

Hair cells of the inner ear are essential for hearing and balance. As a consequence, pathogenic variants in genes specifically expressed in hair cells often cause hereditary deafness. Hair cells are few in number and not easily isolated from the adjacent supporting cells, so the biochemistry and molecular biology of hair cells can be difficult to study. To study gene expression in hair cells, we developed a protocol for hair cell isolation by FACS. With nearly pure hair cells and surrounding cells, from cochlea and utricle and from E16 to P7, we performed a comprehensive cell type-specific RNA-Seq study of gene expression during mouse inner ear development. Expression profiling revealed new hair cell genes with distinct expression patterns: some are specific for vestibular hair cells, others for cochlear hair cells, and some are expressed just before or after maturation of mechanosensitivity. We found that many of the known hereditary deafness genes are much more highly expressed in hair cells than surrounding cells, suggesting that genes preferentially expressed in hair cells are good candidates for unknown deafness genes.

Assessment of functional development of the otolithic system in growing children: a review.

OBJECTIVES: Although the caloric test, rotational test, and posturography have been used to investigate balance function conventionally, and they are older than tests of otolithic organs, yet it seems that most clinicians are less familiar with the development of otolithic (saccular and utricular) function in children. This study reviewed the electrophysiological testing used to assess the functional development of the otolithic system in growing children. METHODS: Based on the literature, studies of cervical vestibular-evoked myogenic potential (cVEMP) and ocular VEMP (oVEMP) tests in children ranging from newborns, small children to adolescents were reviewed. Papers concerning foam posturography in children were also included. RESULTS: The cVEMPs can be elicited in newborns at day 5, whereas the oVEMPs are absent in neonatal period. When children grow to 2 years old, the oVEMPs can be induced with eyes closed condition, while the oVEMPs with eyes up condition can be elicited in children aged >3 years old, with the characteristic parameters similar to adult levels. In contrast with cVEMPs, it is until the neck length >15.3cm (aldolesence), one need not account for neck length in evaluating cVEMP latency. Additionally, foam posturography indicated by the Romberg quotient of the sway velocity/area on foam pad is considered to reflect the otolithic function, which reached adult levels when the children at 12 years old. CONCLUSIONS: For the functional development of the otolithic system in growing children to approach adult levels, the earliest occurrence is the oVEMP test, followed by the foam posturography, and cVEMP test.

A balance of form and function: planar polarity and development of the vestibular maculae.

The mechanosensory hair cells of the inner ear have emerged as one of the primary models for studying the development of planar polarity in vertebrates. Planar polarity is the polarized organization of cells or cellular structures in the plane of an epithelium. For hair cells, planar polarity is manifest at the subcellular level in the polarized organization of the stereociliary bundle and at the cellular level in the coordinated orientation of stereociliary bundles between adjacent cells. This latter organization is commonly called Planar Cell Polarity and has been described in the greatest detail for auditory hair cells of the cochlea. A third level of planar polarity, referred to as tissue polarity, occurs in the utricular and saccular maculae; two inner ear sensory organs that use hair cells to detect linear acceleration and gravity. In the utricle and saccule hair cells are divided between two groups that have opposite stereociliary bundle polarities and, as a result, are able to detect movements in opposite directions. Thus vestibular hair cells are a unique model system for studying planar polarity because polarization develops at three different anatomical scales in the same sensory organ. Moreover the system has the potential to be used to dissect functional interactions between molecules regulating planar polarity at each of the three levels. Here the significance of planar polarity on vestibular system function will be discussed, and the molecular mechanisms associated with development of planar polarity at each anatomical level will be reviewed. Additional aspects of planar polarity that are unique to the vestibular maculae will also be introduced.

Over half the hair cells in the mouse utricle first appear after birth, with significant numbers originating from early postnatal mitotic production in peripheral and striolar growth zones.

Many non-mammalian vertebrates produce hair cells throughout life and recover from hearing and balance deficits through regeneration. In contrast, embryonic production of hair cells declines sharply in mammals where deficits from hair cell losses are typically permanent. Hair cell density estimates recently suggested that the vestibular organs of mice continue to add hair cells after birth, so we undertook comprehensive counting in murine utricles at different ages. The counts show that 51% of the hair cells in adults arise during the 2 weeks after birth. Immature hair cells are most common near the neonatal macula's peripheral edge and striola, where anti-Ki-67 labels cycling nuclei in zones that appear to contain niches for supporting-cell-like stem cells. In vivo lineage tracing in a novel reporter mouse where tamoxifen-inducible supporting cell-specific Cre expression switched tdTomato fluorescence to eGFP fluorescence showed that proteolipid-protein-1-expressing supporting cells are an important source of the new hair cells. To assess the contributions of postnatal cell divisions, we gave mice an injection of BrdU or EdU on the day of birth. The labels were restricted to supporting cells 1 day later, but by 12 days, 31% of the labeled nuclei were in myosin-VIIA-positive hair cells. Thus, hair cell populations in neonatal mouse utricles grow appreciably through two processes: the progressive differentiation of cells generated before birth and the differentiation of new cells arising from divisions of progenitors that progress through S phase soon after birth. Subsequent declines in these processes coincide with maturational changes that appear unique to mammalian supporting cells.

Critical roles of transitional cells and Na/K-ATPase in the formation of vestibular endolymph.

The mechanotransduction of vestibular sensory cells depends on the high endolymphatic potassium concentration ([K+]) maintained by a fine balance between K+ secretion and absorption by epithelial cells. Despite the crucial role of endolymph as an electrochemical motor for mechanotransduction, little is known about the processes that govern endolymph formation. To address these, we took advantage of an organotypic rodent model, which regenerates a genuine neonatal vestibular endolymphatic compartment, facilitating the determination of endolymphatic [K+] and transepithelial potential (Vt) during endolymph formation. While mature Vt levels are almost immediately achieved, K+ accumulates to reach a steady [K+] by day 5 in culture. Inhibition of sensory cell K+ efflux enhances [K+] regardless of the blocker used (FM1.43, amikacin, gentamicin, or gadolinium). Targeting K+ secretion with bumetanide partially and transiently reduces [K+], while ouabain application and Kcne1 deletion almost abolishes it. Immunofluorescence studies demonstrate that dark cells do not express Na-K-2Cl cotransporter 1 (the target of bumetanide) in cultured and young mouse utricles, while Na/K-ATPase (the target of ouabain) is found in dark cells and transitional cells. This global analysis of the involvement of endolymphatic homeostasis actors in the immature organ (1) confirms that KCNE1 channels are necessary for K+ secretion, (2) highlights Na/K-ATPase as the key endolymphatic K+ provider and shows that Na-K-2Cl cotransporter 1 has a limited impact on K+ influx, and (3) demonstrates that transitional cells are involved in K+ secretion in the early endolymphatic compartment.

Ontogeny of auditory saccular sensitivity in the plainfin midshipman fish, Porichthys notatus.

The auditory system of the plainfin midshipman fish, Porichthys notatus, is an important sensory receiver system used to encode intraspecific social communication signals in adults, but the response properties and function of this receiver system in pre-adult stages are less known. In this study we examined the response properties of auditory-evoked potentials from the midshipman saccule, the main organ of hearing in this species, to determine whether the frequency response and auditory threshold of saccular hair cells to behaviorally relevant single tone stimuli change during ontogeny. Saccular potentials were recorded from three relative sizes of midshipman fish: small juveniles [1.9-3.1 cm standard length (SL), large juveniles (6.8-8.0 cm SL) and non-reproductive adults (9.0-22.6 cm SL)]. The auditory evoked potentials were recorded from the rostral, middle and caudal regions of the saccule while single tone stimuli (75-1,025 Hz) were presented via an underwater speaker. We show that the frequency response and auditory threshold of the midshipman saccule is established early in development and retained throughout ontogeny. We also show that saccular sensitivity to frequencies greater than 385 Hz increases with age/size and that the midshipman saccule of small and large juveniles, like that of non-reproductive adults, is best suited to detect low frequency sounds (<105 Hz) in their natural acoustic environment.

Expression of PINK1 in the brain, eye and ear of mouse during embryonic development.

PINK1 is a 581 amino acid protein with a serine/threonine kinase domain and an N-terminal mitochondrial targeting motif. The enzyme is expressed in the brain as well as in several tissues such as heart, skeletal muscle, liver, kidney, pancreas and testis. In the present study, we have investigated by Western blot analysis and immunohistochemistry the presence and distribution of PINK1 in the brain, eye and inner ear of mouse during embryonic development. In the brain we detected two PINK1 molecular isoforms of 55 kDa and 66 kDa. Immunoreactive perikarya first appeared at stage E15 in the diencephalon within the thalamus, the hypothalamus, the periventricular layers of the third ventricle and in the rhombencephalon at level of the pons. Subsequently, new PINK1-positive neurons were found in the midbrain within the floor and the periventricular layers of the ventral wall of the mesencephalic vesicle (stage E17) as well as in the neopallial cortex, the tegmentum of the midbrain and the periventricular region of the caudal part of the rhombencephalon (stage E19). At P0, PINK1-immunoreactive cells appeared in the striatum, the mantle layer and caudal part of the medulla oblongata and the cerebellum. The spatio-temporal expression of PINK1 and its heterogeneous distribution suggest that the enzyme might be involved in neuroregulatory processes during embryogenesis. In the eye, PINK1-immunoreactivity was found in the lens and in the cornea, whereas in the inner ear the enzyme was expressed in the ependymal and subependymal cells of the saccule and in the semicircular canals indicating that PINK1 plays a role in the development of these sensory organs.

Thyroid hormone mediates otolith growth and development during flatfish metamorphosis.

Flatfish begin life as bilaterally symmetrical larvae that swim up-right, then abruptly metamorphose into asymmetrically shaped juveniles with lateralized swimming postures. Flatfish metamorphosis is mediated entirely by thyroid hormone (TH). Changes in flatfish swim posture are thought to be regulated via vestibular remodeling, although the influence of TH on teleost inner ear development remains unclear. This study addresses the role of TH on the development of the three otolith end-organs (sacculus, utricle, and lagena) during southern flounder (Paralichthys lethostigma) metamorphosis. Compared with pre-metamorphosis, growth rates of the sacculus and utricle otoliths increase dramatically during metamorphosis in a manner that is uncoupled from general somatic growth. Treatment of P. lethostigma larvae with methimazol (a pharmacological inhibitor of endogenous TH production) inhibits growth of the sacculus and utricle, whereas treatment with TH dramatically accelerates their growth. In contrast with the sacculus and utricle otoliths that begin to form and mineralize during embryogenesis, a non-mineralized lagena otolith is first visible 10-12 days after hatching. The lagena grows during pre- and pro-metamorphosis, then abruptly mineralizes during metamorphic climax. Mineralization of the lagena, but not growth, can be induced with TH treatment, whereas treatment with methimazol completely inhibits lagena mineralization without inhibiting its growth. These findings suggest that during southern flounder metamorphosis TH exerts differential effects on growth and development among the three types of otolith.

Development of otolith receptors in Japanese quail.

This study examined the morphological development of the otolith vestibular receptors in quail. Here, we describe epithelial growth, hair cell density, stereocilia polarization, and afferent nerve innervation during development. The otolith maculae epithelial areas increased exponentially throughout embryonic development reaching asymptotic values near posthatch day P7. Increases in hair cell density were dependent upon macular location; striolar hair cells developed first followed by hair cells in extrastriola regions. Stereocilia polarization was initiated early, with defining reversal zones forming at E8. Less than half of all immature hair cells observed had nonpolarized internal kinocilia with the remaining exhibiting planar polarity. Immunohistochemistry and neural tracing techniques were employed to examine the shape and location of the striolar regions. Initial innervation of the maculae was by small fibers with terminal growth cones at E6, followed by collateral branches with apparent bouton terminals at E8. Calyceal terminal formation began at E10; however, no mature calyces were observed until E12, when all fibers appeared to be dimorphs. Calyx afferents innervating only Type I hair cells did not develop until E14. Finally, the topographic organization of afferent macular innervation in the adult quail utricle was quantified. Calyx and dimorph afferents were primarily confined to the striolar regions, while bouton fibers were located in the extrastriola and Type II band. Calyx fibers were the least complex, followed by dimorph units. Bouton fibers had large innervation fields, with arborous branches and many terminal boutons.

Dominant-negative inhibition of M-like potassium conductances in hair cells of the mouse inner ear.

Sensory hair cells of the inner ear express multiple physiologically defined conductances, including mechanotransduction, Ca(2+), Na(+), and several distinct K(+) conductances, all of which are critical for normal hearing and balance function. Yet, the molecular underpinnings and their specific contributions to sensory signaling in the inner ear remain obscure. We sought to identify hair-cell conductances mediated by KCNQ4, which, when mutated, causes the dominant progressive hearing loss DFNA2. We used the dominant-negative pore mutation G285S and packaged the coding sequence of KCNQ4 into adenoviral vectors. We transfected auditory and vestibular hair cells of organotypic cultures generated from the postnatal mouse inner ear. Cochlear outer hair cells and vestibular type I cells that expressed the transfection marker, green fluorescent protein, and the dominant-negative KCNQ4 construct lacked the M-like conductances that typify nontransfected control hair cells. As such, we conclude that the M-like conductances in mouse auditory and vestibular hair cells can include KCNQ4 subunits and may also include KCNQ4 coassembly partners. To examine the function of M-like conductances in hair cells, we recorded from cells transfected with mutant KCNQ4 and injected transduction current waveforms in current-clamp mode. Because the M-like conductances were active at rest, they contributed to the very low potassium-selective input resistance, which in turn hyperpolarized the resting potential and significantly attenuated the amplitude of the receptor potential. Modulation of M-like conductances may allow hair cells the ability to control the amplitude of their response to sensory stimuli.

Differential distribution of stem cells in the auditory and vestibular organs of the inner ear.

The adult mammalian cochlea lacks regenerative capacity, which is the main reason for the permanence of hearing loss. Vestibular organs, in contrast, replace a small number of lost hair cells. The reason for this difference is unknown. In this work we show isolation of sphere-forming stem cells from the early postnatal organ of Corti, vestibular sensory epithelia, the spiral ganglion, and the stria vascularis. Organ of Corti and vestibular sensory epithelial stem cells give rise to cells that express multiple hair cell markers and express functional ion channels reminiscent of nascent hair cells. Spiral ganglion stem cells display features of neural stem cells and can give rise to neurons and glial cell types. We found that the ability for sphere formation in the mouse cochlea decreases about 100-fold during the second and third postnatal weeks; this decrease is substantially faster than the reduction of stem cells in vestibular organs, which maintain their stem cell population also at older ages. Coincidentally, the relative expression of developmental and progenitor cell markers in the cochlea decreases during the first 3 postnatal weeks, which is in sharp contrast to the vestibular system, where expression of progenitor cell markers remains constant or even increases during this period. Our findings indicate that the lack of regenerative capacity in the adult mammalian cochlea is either a result of an early postnatal loss of stem cells or diminishment of stem cell features of maturing cochlear cells.