Stereocilia Rootlets: Actin-Based Structures That Are Essential for Structural Stability of the Hair Bundle.

Sensory hair cells of the inner ear rely on the hair bundle, a cluster of actin-filled stereocilia, to transduce auditory and vestibular stimuli into electrical impulses. Because they are long and thin projections, stereocilia are most prone to damage at the point where they insert into the hair cell's soma. Moreover, this is the site of stereocilia pivoting, the mechanical movement that induces transduction, which additionally weakens this area mechanically. To bolster this fragile area, hair cells construct a dense core called the rootlet at the base of each stereocilium, which extends down into the actin meshwork of the cuticular plate and firmly anchors the stereocilium. Rootlets are constructed with tightly packed actin filaments that extend from stereocilia actin filaments which are wrapped with TRIOBP; in addition, many other proteins contribute to the rootlet and its associated structures. Rootlets allow stereocilia to sustain innumerable deflections over their lifetimes and exemplify the unique manner in which sensory hair cells exploit actin and its associated proteins to carry out the function of mechanotransduction.

Functional Analysis of the Transmembrane and Cytoplasmic Domains of Pcdh15a in Zebrafish Hair Cells.

Protocadherin 15 (PCDH15) is required for mechanotransduction in sensory hair cells as a component of the tip link. Isoforms of PCDH15 differ in their cytoplasmic domains (CD1, CD2, and CD3), but share the extracellular and transmembrane (TMD) domains, as well as an intracellular domain known as the common region (CR). In heterologous expression systems, both the TMD and CR of PCDH15 have been shown to interact with members of the mechanotransduction complex. The in vivo significance of these protein-protein interaction domains of PCDH15 in hair cells has not been determined. Here, we examined the localization and function of the two isoforms of zebrafish Pcdh15a (CD1 and CD3) in pcdh15a-null mutants by assessing Pcdh15a transgene-mediated rescue of auditory/vestibular behavior and hair cell morphology and activity. We found that either isoform alone was able to rescue the Pcdh15a-null phenotype and that the CD1- or CD3-specific regions were dispensable for hair bundle integrity and labeling of hair cells with FM4-64, which was used as a proxy for mechanotransduction. When either the CR or TMD domain was deleted, the mutated proteins localized to the stereocilial tips, but were unable to rescue FM4-64 labeling. Disrupting both domains led to a complete failure of Pcdh15a to localize to the hair bundle. Our findings demonstrate that the TMD and cytoplasmic CR domains are required for the in vivo function of Pcdh15a in zebrafish hair cells.SIGNIFICANCE STATEMENT Tip links transmit force to mechanotransduction channels at the tip of hair bundles in sensory hair cells. One component of tip links is Protocadherin 15 (PCDH15). Here, we demonstrate that, when transgenically expressed, either zebrafish Pcdh15a-cytodomain 1 (CD1) or Pcdh15a-CD3 can rescue the phenotype of a pcdh15a-null mutant. Even when lacking the specific regions for CD1 or CD3, truncated Pcdh15a that contains the so-called common region (CR) at the cytoplasmic/membrane interface still has the ability to rescue similar to full-length Pcdh15a. In contrast, Pcdh15a lacking the entire cytoplasmic domain is not functional. These results demonstrate that the CR plays a key role in the mechanotransduction complex in hair cells.

Biomechanics of hair cell kinocilia: experimental measurement of kinocilium shaft stiffness and base rotational stiffness with Euler-Bernoulli and Timoshenko beam analysis.

Vestibular hair cell bundles in the inner ear contain a single kinocilium composed of a 9+2 microtubule structure. Kinocilia play a crucial role in transmitting movement of the overlying mass, otoconial membrane or cupula to the mechanotransducing portion of the hair cell bundle. Little is known regarding the mechanical deformation properties of the kinocilium. Using a force-deflection technique, we measured two important mechanical properties of kinocilia in the utricle of a turtle, Trachemys (Pseudemys) scripta elegans. First, we measured the stiffness of kinocilia with different heights. These kinocilia were assumed to be homogenous cylindrical rods and were modeled as both isotropic Euler-Bernoulli beams and transversely isotropic Timoshenko beams. Two mechanical properties of the kinocilia were derived from the beam analysis: flexural rigidity (EI) and shear rigidity (kGA). The Timoshenko model produced a better fit to the experimental data, predicting EI=10,400 pN µm(2) and kGA=247 pN. Assuming a homogenous rod, the shear modulus (G=1.9 kPa) was four orders of magnitude less than Young's modulus (E=14.1 MPa), indicating that significant shear deformation occurs within deflected kinocilia. When analyzed as an Euler-Bernoulli beam, which neglects translational shear, EI increased linearly with kinocilium height, giving underestimates of EI for shorter kinocilia. Second, we measured the rotational stiffness of the kinocilium insertion (κ) into the hair cell's apical surface. Following BAPTA treatment to break the kinocilial links, the kinocilia remained upright, and κ was measured as 177±47 pN µm rad(-1). The mechanical parameters we quantified are important for understanding how forces arising from head movement are transduced and encoded by hair cells.

The septate junction protein caspr is required for structural support and retention of KCNQ4 at calyceal synapses of vestibular hair cells.

The afferent innervation contacting the type I hair cells of the vestibular sensory epithelia form distinct calyceal synapses. The apposed presynaptic and postsynaptic membranes at this large area of synaptic contact are kept at a remarkably regular distance. Here, we show by freeze-fracture electron microscopy that a patterned alignment of proteins at the calyceal membrane resembles a type of intercellular junction that is rare in vertebrates, the septate junction (SJ). We found that a core molecular component of SJs, Caspr, colocalizes with the K(+) channel KCNQ4 at the postsynaptic membranes of these calyceal synapses. Immunolabeling and ultrastructural analyses of Caspr knock-out mice reveal that, in the absence of Caspr, the separation between the membranes of the hair cells and the afferent neurons is conspicuously irregular and often increased by an order of magnitude. In these mutants, KCNQ4 fails to cluster at the postsynaptic membrane and appears diffused along the entire calyceal membrane. Our results indicate that a septate-like junction provides structural support to calyceal synaptic contact with the vestibular hair cell and that Caspr is required for the recruitment or retention of KCNQ4 at these synapses.

Single-cell proteomics reveals changes in expression during hair-cell development.

Hearing and balance rely on small sensory hair cells that reside in the inner ear. To explore dynamic changes in the abundant proteins present in differentiating hair cells, we used nanoliter-scale shotgun mass spectrometry of single cells, each ~1 picoliter, from utricles of embryonic day 15 chickens. We identified unique constellations of proteins or protein groups from presumptive hair cells and from progenitor cells. The single-cell proteomes enabled the de novo reconstruction of a developmental trajectory using protein expression levels, revealing proteins that greatly increased in expression during differentiation of hair cells (e.g., OCM, CRABP1, GPX2, AK1, GSTO1) and those that decreased during differentiation (e.g., TMSB4X, AGR3). Complementary single-cell transcriptome profiling showed corresponding changes in mRNA during maturation of hair cells. Single-cell proteomics data thus can be mined to reveal features of cellular development that may be missed with transcriptomics.

High frequency of the IVS2-2A>G DNA sequence variation in SLC26A5, encoding the cochlear motor protein prestin, precludes its involvement in hereditary hearing loss.

BACKGROUND: Cochlear outer hair cells change their length in response to variations in membrane potential. This capability, called electromotility, is believed to enable the sensitivity and frequency selectivity of the mammalian cochlea. Prestin is a transmembrane protein required for electromotility. Homozygous prestin knockout mice are profoundly hearing impaired. In humans, a single nucleotide change in SLC26A5, encoding prestin, has been reported in association with hearing loss. This DNA sequence variation, IVS2-2A>G, occurs in the exon 3 splice acceptor site and is expected to abolish splicing of exon 3. METHODS: To further explore the relationship between hearing loss and the IVS2-2A>G transition, and assess allele frequency, genomic DNA from hearing impaired and control subjects was analyzed by DNA sequencing. SLC26A5 genomic DNA sequences from human, chimp, rat, mouse, zebrafish and fruit fly were aligned and compared for evolutionary conservation of the exon 3 splice acceptor site. Alternative splice acceptor sites within intron 2 of human SLC26A5 were sought using a splice site prediction program from the Berkeley Drosophila Genome Project. RESULTS: The IVS2-2A>G variant was found in a heterozygous state in 4 of 74 hearing impaired subjects of Hispanic, Caucasian or uncertain ethnicity and 4 of 150 Hispanic or Caucasian controls (p = 0.45). The IVS2-2A>G variant was not found in 106 subjects of Asian or African American descent. No homozygous subjects were identified (n = 330). Sequence alignment of SLC26A5 orthologs demonstrated that the A nucleotide at position IVS2-2 is invariant among several eukaryotic species. Sequence analysis also revealed five potential alternative splice acceptor sites in intron 2 of human SLC26A5. CONCLUSION: These data suggest that the IVS2-2A>G variant may not occur more frequently in hearing impaired subjects than in controls. The identification of five potential alternative splice acceptor sites in intron 2 of human SLC26A5 suggests a potential mechanism by which expression of prestin might be maintained in cells carrying the SLC26A5 IVS2-2A>G DNA sequence variation. Additional studies are needed to evaluate the effect of the IVS2-2A>G transition on splicing of SLC26A5 transcripts and characterize the hearing status of individuals homozygous for the IVS2-2A>G variant.

Molecular characterization of an inward rectifier channel (IKir) found in avian vestibular hair cells: cloning and expression of pKir2.1.

A fast inwardly rectifying current has been observed in some of the sensory cells (hair cells) of the inner ear of several species. While the current was presumed to be an IKir current, contradictory evidence existed as to whether the cloned channel actually belonged to the Kir2.0 subfamily of potassium inward rectifiers. In this paper, we report for the first time converging evidence from electrophysiological, biochemical, immunohistochemical, and genetic studies that show that the Kir2.1 channel carries the fast inwardly rectifying currents found in pigeon vestibular hair cells. Following cytoplasm extraction from single type II and multiple pigeon vestibular hair cells, mRNA was reverse transcribed, amplified, and sequenced. The open reading frame (ORF), consisting of a 1,284-bp nucleotide sequence, showed 94, 85, and 83% identity with Kir2.1 subunit sequences from chick lens, Kir2 sequences from human heart, and a mouse macrophage cell line, respectively. Phylogenetic analyses revealed that pKir2.1 formed an immediate node with hKir2.1 but not with hKir2.2-2.4. Hair cells (type I and type II) and supporting cells in the sensory epithelium reacted positively with a Kir2.1 antibody. The whole cell current recorded in oocytes and CHO cells, transfected with pigeon hair cell Kir2.1 (pKir2.1), demonstrated blockage by Ba2+ and sensitivity to changing K+ concentration. The mean single-channel linear slope conductance in transfected CHO cells was 29 pS. The open dwell time was long (approximately 300 ms at -100 mV), and the closed dwell time was short (approximately 34 ms at -100 mV). Multistates ranging from 3-6 were noted in some single-channel responses. All of the above features have been described for other Kir2.1 channels. Current clamp studies of native pigeon vestibular hair cells illustrated possible physiological roles of the channel and showed that blockage of the channel by Ba2+ depolarized the resting membrane potential by approximately 30 mV. Negative currents hyperpolarized the membrane approximately 20 mV before block but approximately 60 mV following block. RT-PCR studies revealed that the pKir2.1 channels found in pigeon vestibular hair cells were also present in pigeon vestibular nerve, vestibular ganglion, lens, neck muscle, brain (brain stem, cerebellum and optic tectum), liver, and heart.

Ultrastructural analysis of [3H]thymidine-labeled cells in the rat utricular macula.

Ototoxic drugs stimulate cell proliferation in adult rat vestibular sensory epithelia, as does the infusion of transforming growth factor alpha (TGFalpha) plus insulin. We sought to determine whether new hair cells can be regenerated by means of a mitotic pathway. Previously, studies have shown that the nuclei of some newly generated cells are located in the lumenal half of the sensory epithelium, suggesting that some may be newly generated sensory hair cells. The aim of this study was to examine the ultrastructural characteristics of newly proliferated cells after TGFalpha stimulation and/or aminoglycoside damage in the utricular sensory epithelium of the adult rat. The cell proliferation marker tritiated-thymidine was infused, with or without TGFalpha plus insulin, into the inner ears of normal or aminoglycoside-damaged rats for 3 or 7 days by means of osmotic pumps. Autoradiographic techniques and light microscopy were used to identify cells synthesizing DNA. Sections with labeled cells were re-embedded, processed for transmission electron microscopy, and the ultrastructural characteristics of the labeled cells were examined. The following five classes of tritiated-thymidine labeled cells were identified in the sensory epithelium: (1) labeled cells with synaptic specializations that appeared to be newly generated hair cells, (2) labeled supporting cells, (3) labeled leukocytes, (4) labeled cells that we have classified as "active cells" in that they are relatively nondescript but contain massive numbers of polyribosomes, and (5) labeled degenerating hair cells. These findings suggest that new hair cells can be generated in situ by means of a mitotic mechanism in the vestibular sensory epithelium of adult mammals.

Nitric oxide synthase localized in a subpopulation of vestibular efferents with NADPH diaphorase histochemistry and nitric oxide synthase immunohistochemistry.

Efferent innervation of the vestibular labyrinth is known to be cholinergic. More recent studies have also demonstrated the presence of the neuropeptide calcitonin gene-related peptide in this system. Nitric oxide is one of a new class of neurotransmitters, the gaseous transmitters. It acts as a second messenger and neurotransmitter in diverse physiological systems. We decided to investigate the anatomical distribution of the synthetic enzyme for nitric oxide, nitric oxide synthase (NOS), to clarify the role of nitric oxide in the vestibular periphery. NADPH diaphorase histochemical and NOS I immunohistochemical studies were done in the adult chinchilla and rat vestibular brainstem; diaphorase histochemistry was done in the chinchilla periphery. Retrograde tracing studies to verify the presence of NOS in brainstem efferent neurons were performed in young chinchillas. Our light microscopic results show that NOS I, as defined mainly by the presence of NADPH diaphorase, is present in a subpopulation of both brainstem efferent neurons and peripheral vestibular efferent boutons. Our ultrastructural results confirm these findings in the periphery. NADPH diaphorase is also present in a subpopulation of type I hair cells, suggesting that nitric oxide might be produced in and act locally upon these cells and other elements in the sensory epithelium. A hypothesis about how nitric oxide is produced in the vestibular periphery and how it may interact with other elements in the vestibular sensory apparatus is presented in the discussion.

Differential subcellular immunolocalization of voltage-gated calcium channel alpha1 subunits in the chinchilla cristae ampullaris.

The immunohistochemical localization of alpha1A, alpha1B, alpha1C, alpha1D and alpha1E voltage-gated calcium channel subunits was investigated in the chinchilla cristae ampullaris and Scarpa's ganglia at the light and electron microscopy level with the use of specific antipeptide antibodies directed against these subunits. The stereocilia membrane of type I and type II hair cells was immunoreactive for alpha1B along its entire length. The basolateral membrane of both types of hair cells was alpha1B, alpha1C and alpha1D immunoreactive. Neurons in the Scarpa's ganglia and afferent nerve terminals in the cristae were immunoreactive for alpha1C and alpha1B. No specific immunoreactivity to alpha1A or alpha1E was seen in the sensory epithelia or ganglia. These findings are consistent with the presence of alpha1B (N-type channel), alpha1C and alpha1D (L-type channels) in the vestibular hair cells, and alpha1B (N-type channel) and alpha1C (L-type channel) in primary vestibular neurons.

Isolation of A-type K+ current in hair cells of the frog crista ampullaris.

Different procedures to isolate the K+ A-type current (IA) from other membrane currents were tested on the complex inactivating outward K+ current generated in hair cells from the peripheral regions of the frog crista ampullaris. Experiments were performed in thin slices of epithelium using the whole-cell configuration of the patch-clamp technique. The conventional conditioning voltage protocol did not allow a satisfactory isolation of IA, due to the presence of other K+ currents showing overlapping steady-state inactivation properties. An attempt to block other K+ currents using calcium-free saline containing 50 mM TEA also failed to provide a satisfactory isolation of IA, due to contamination by a residual sustained current, probably consisting of a slow delayed outward K+ current (IK). Use of the selective A-channel blocker 4-aminopyridine (4-AP) at concentrations < 12 mM was also unsatisfactory because at these concentrations 4-AP produced a voltage-dependent blockade. Conversely, use of 4-AP at concentrations of 15-20 mM allowed a good separation of an uncontaminated IA. These results indicate that IA in hair cells of vestibular epithelium can be isolated most effectively by the 4-AP procedure, provided that sufficiently high concentrations of the A-channel blocker are used.

Subcellular immunolocalization of NMDA receptor subunit NR-1 in the chinchilla vestibular periphery.

The immunohistochemical localization of N-methyl-D-aspartate (NMDA) glutamate receptor subunit, NR-1 was investigated in the chinchilla cristae ampullaris and utricular maculae at the light and electron microscopy level with the use of specific antipeptide antibodies. The afferent calyces that innervate type I hair cell, and the basolateral type I vestibular hair cell is NR-1 immunoreactive. The afferent boutons innervating type II hair cells and the basal portion of type II hair cell are NR-1 non-immunoreactive. These findings are consistent with NMDA receptor mediation of afferent excitatory neurotransmission from type I, but not type II hair cells to the primary afferent vestibular nerve. The NMDA receptors on the type I hair cell are located in areas of synaptic specialization, and may play a role in autoregulation. The localization of the NMDA receptor subunit in type I but not type II hair cells is intriguing.

Differential expression of alpha2-7, alpha9 and beta2-4 nicotinic acetylcholine receptor subunit mRNA in the vestibular end-organs and Scarpa's ganglia of the rat.

To further characterize the pattern of expression of the nicotinic acetylcholine receptor (nAChR) subunits in the peripheral vestibular system, we conducted RT-PCR of all known mammalian nAChR alpha and beta subunits in mRNA extracted from adult rat vestibular primary afferent neurons (Scarpa's ganglia) and vestibular end-organs. Transcripts encoding the alpha2-7 and beta2-4 nAChR subunits were found in the vestibular ganglia, while alpha3, alpha5-7, alpha9 and beta2-4 nAChR subunits were expressed in the vestibular end-organs. These results support previous electrophysiological, immunocytochemical and molecular biological data, and also provide a more complete understanding of the role of nAChRs in the neurochemical transmission subserving the efferent-afferent interaction in the vestibular periphery.

Autoradiographic demonstration of quinuclidinyl benzilate binding sites in the vestibular organs of the gerbil.

Gerbil vestibular tissues were isolated by microdissection and incubated in vitro with 3H-quinuclidinyl benzilate (3H-QNB). Control tissues were incubated in medium containing unlabeled atropine to differentiate non-specific from specific binding. Autoradiographic grain densities were determined by morphometric techniques and evaluated by two-tailed t-test. The label densities of sensory epithelia from experimental preparations of ampulla, utricle and saccule were found to be significantly higher than those in the adjacent endolymphatic compartment and also higher than those of adjacent stromal tissue comprising connective tissue, nerve fibers and capillaries. In contrast, no tissue region in atropine controls showed label density significantly above that of the endolymphatic compartment. Label density of ampullar sensory epithelium incubated with 3H-QNB alone was significantly higher than that of sensory epithelium from utricle or saccule. Grain density was greater in the peripheral regions of the ampullar crista compared to the vertex. Appreciable label was also present in nerve bundles beneath the sensory epithelium of the ampulla. The current study demonstrates the existence of putative muscarinic neurotransmitter/neuromodulator receptor sites in mammalian vestibular sense organs at locations corresponding to efferent innervation, with particularly significant concentrations in the ampulla.

Cellular and subcellular localization of AMPA-selective glutamate receptors in the mammalian peripheral vestibular system.

The cellular and subcellular distribution of AMPA-selective glutamate receptors in the mammalian peripheral vestibular system was examined using antibodies against peptides corresponding to the C-terminal portions of AMPA receptor subunits: GluR1, GluR2/R3 and GluR4. The light and electron microscopic immunocytochemical studies were carried out on Vibratome sections of rat and guinea pig vestibular sensory epithelial and ganglia. In the epithelium, GluR1 subunit immunoreactivity appeared as accumulations of patches outlining the baso-lateral periphery of the type I sensory cells. The GluR1-immunoreactive microareas were postsynaptically distributed on the membranes of calyceal afferent fibers. GluR2/R3 immunoreactivity was present in the sensory cells. GluR4 was not detected. In the vestibular ganglion, the neurons were densely stained with antibodies to GluR2/R3 and GluR4. The fibroblasts and the Schwann cells were also intensely stained with antibodies to GluR2/R3 and GluR4. In the sensory cells, the AMPA receptors, GluR2/R3, may function as (1) autoreceptors controlling afferent neurotransmitter release or (2) 'postsynaptic' receptors activated by the neurotransmitter release of the afferent calyx. The detection of GluR1 at postsynaptic sites in the afferent fibers provides anatomical evidence for the role of glutamate as a neurotransmitter of sensory cells. In the ganglion neurons, GluR2/R3 and GluR4 may represent reserve intracytoplasmic pools of receptor subunits in transit to the postsynaptic sites. In the Schwann cells, GluR2/R3 and GluR4 may be involved in neuronal-glial signalling at the nodes of Ranvier.

The combined effects of trkB and trkC mutations on the innervation of the inner ear.

Previous research has demonstrated that only the two neurotrophins and their cognate receptors are necessary for the support of the inner ear innervation. However, detailed analyses of patterns of innervation in various combinations of neurotrophin receptor mutants are lacking. We provide here such an analysis of the distribution of afferent and efferent fibers to the ear in various combinations of neurotrophin receptor mutants using the lipophilic tracer Dil. In the vestibular system, trkC+/- heterozygosity aggravates the trkB-/- mutation effect and causes almost complete loss of vestibular neurons. In the cochlea innervation, various mutations are each characterized by specific topological absence of spiral neurons in Rosenthal's canal of the cochlea. trkC-/- mutation alone or in combination with trkB+/- heterozygosity causes absence of all basal turn spiral neurons and afferent fibers extend from the middle turn to the basal turn along inner hair cells with little or no contribution to outer hair cells. Both types of basal turn spiral neurons appear to develop and project via radial fibers to inner and, more sparingly, outer hair cells. Simple trkB-/- mutations show a reduction of fibers to outer hair cells in the apex and, less obvious, in the basal turn. Basal turn spiral neurons may be the only neurons present at birth in the cochlea of a trkB-/- mutant mouse combined with trkC+/- heterozygosity. In addition, the trkB-/- mutation combined with trkC+/- heterozygosity has a patchy and variable loss of middle turn spiral neurons in mice of different litters. Comparisons of patterns of innervation of afferent and efferent fibers show a striking similarity of absence of fibers to topologically corresponding areas. For example, in trkC-/- mutants afferents reach the basal turn, spiraling along the cochlea, rather than through radial fibers and efferent fibers follow the same pathway rather than emanating from intraganglionic spiral fibers. The data presented suggest that there are regional specific effects with some bias towards a specific spiral ganglion type: trkC is essential for support of basal turn spiral neurons whereas trkB appears to be more important for middle and apical turn spiral neurons.

Subcellular innervation patterns of the calcitonin gene-related peptidergic efferent terminals in the chinchilla vestibular periphery.

We examined the ultrastructural distribution of calcitonin gene-related peptide immunoreactivity in the peripheral vestibular system of the chinchilla to study the innervation patterns of this efferent neuropeptide. Immunoelectron microscopic localization of calcitonin gene-related peptide immunoreactive terminals in the maculae and cristae revealed an extensive innervation pattern on the afferent vestibular pathway. Calcitonin gene-related peptide immuno-reactive terminals made synaptic contacts with the unmyelinated portions of the primary afferent vestibular dendrites innervating both type I and type II hair cells. Abundant synaptic contact between calcitonin gene-related peptide immunoreactive terminals and the chalices surrounding type I hair cells was observed. Direct contact between calcitonin gene-related peptide immunoreactive terminals and type II hair cells was observed. In addition, vesiculated efferent terminals without calcitonin gene-related peptide immunoreactivity were seen synapsing on the chalices of type II hair cells and on the surrounding type I hair cells. The primary afferent somata in the vestibular ganglion of Scarpa did not contain calcitonin gene-related peptide immunoreactivity. Unmyelinated calcitonin gene-related peptide immunoreactive axons passed among the primary afferent fibers in Scarpa's ganglion, and these fibers continued through the subepithelial regions of the vestibular end-organs. The calcitonin gene-related peptide immunoreactive axons ramified to produce numerous calcitonin gene-related peptide immunoreactive terminals throughout the neurosensory epithelium of the maculae and cristae. These data suggest that calcitonin gene-related peptide-mediated modulation of the afferent vestibular system is functionally important.

Alpha and beta subunits of acetylcholine receptors in the human inner ear.

The localization and distribution of nicotinic acetylcholine receptors (n-ACh-r) was characterized by studying alpha and beta subunits in the adult human inner ear by FITC fluorescence technique. In the cochlea, distinct fluorescence staining occurred for beta subunits in outer hair cells (OHCs), but no alpha subunits were identified. Beta subunits differ quantitatively between the three rows of OHCs, decreasing along a base-to-apex gradient in the cochlea. Both alpha and beta subunits were identified on spiral ganglion cells, adjacent nerve fibres and in vestibular hair cells (HCs). It would appear that they form an active complex in n-ACh-r at these locations.

Vestibular hair cells of the chick express the nicotinic acetylcholine receptor subunit alpha 9.

The efferent cholinergic pathways to the vestibular periphery have yet to be fully characterized. While the nicotinic acetylcholine receptor subunit (nAChR) alpha 9 is now regarded as the principle receptor for efferent cholinergic signaling to the organ of Corti, there is still uncertainty over how the more complex efferent effects of the labyrinth are produced. Recent experimental work has demonstrated that the nAChR alpha 9 is present in the vestibular end-organs of the rat and mouse, suggesting that alpha 9 may be one of the mediators of efferent cholinergic signaling in the vestibular periphery as well. In this experiment, we sought to determine whether alpha 9 was also present in the vestibular end-organs of the chick. A homologue of alpha 9 has been cloned recently from the chick cochlea. Using reverse transcription polymerase chain reaction (RT-PCR), individual vestibular end-organ preparations, including posterior ampulla, combined horizontal and superior ampulla, saccule, utricle, and the vestibular ganglion were screened for alpha 9 messenger RNA expression. In each end-organ and the vestibular ganglion, a cDNA of the expected size was obtained by RT-PCR and was confirmed to be alpha 9 by sequence analysis. Further, alpha 9 mRNA was identified by RT-PCR from individually isolated type I and type II vestibular hair cells (single-cell RT-PCR). Lastly, insitu hybridization using digoxigenin-labeled alpha 9 riboprobes confirmed the presence of alpha 9 in type I and type II hair cells throughout the vestibular periphery. These results demonstrate the expression of alpha 9 in the vestibular end-organs of the chick, and lend further support for the role of alpha 9 as a mediator of efferent cholinergic signaling in vestibular hair cells.