Age-related changes in the number of cresyl-violet-stained, parvalbumin and NMDAR 2B expressing neurons in the human spiral ganglion.

Animal-studies associate age-related hearing loss (presbycusis) with decreasing number of spiral ganglion neurons (SGNs) in Rosenthal's canal (RC) of cochlea. The excitatory neurotransmitter for SGNs is glutamate (through its receptor NMDAR 2B), which can be neurotoxic through Ca2+ overload. Neurotoxicity is balanced by calcium-binding proteins (CBPs) like Parvalbumin (PV), which is the predominant CBP of the SGNs. To estimate the volume of the RC and total number of SGNs that are immunoreactive to PV and NMDAR 2B, we used unbiased stereology in 35 human cochleae derived from cadavers of persons from 2nd to 8th decade of life (subsequently statistically divided into two groups) and compared them to the total number of cresyl violet (CV) stained SGNs. We also estimated the volume of individual neurons and their nuclei. Regression analysis was made on estimated parameters against age. Hierarchical-cluster analysis was done on the neuronal against neuronal nuclear volumes.The average volume of the RC did not change with increasing age (p = 0.4115). The total number of SGNs (CV-stained and those separately expressing PV and NMDAR 2B) significantly decreased with age (p < 0.001). We identified three distinct populations of neurons on the basis of their volumes among SGNs. Thus, there is significant age-related decline in the total number of SGNs, which starts early in life. It may be due to ambient noise and inadequate neutralisation of excitotoxicity.

Homeostatic Control of Spontaneous Activity in the Developing Auditory System.

Neurons in the developing auditory system exhibit spontaneous bursts of activity before hearing onset. How this intrinsically generated activity influences development remains uncertain, because few mechanistic studies have been performed in vivo. We show using macroscopic calcium imaging in unanesthetized mice that neurons responsible for processing similar frequencies of sound exhibit highly synchronized activity throughout the auditory system during this critical phase of development. Spontaneous activity normally requires synaptic excitation of spiral ganglion neurons (SGNs). Unexpectedly, tonotopic spontaneous activity was preserved in a mouse model of deafness in which glutamate release from hair cells is abolished. SGNs in these mice exhibited enhanced excitability, enabling direct neuronal excitation by supporting cell-induced potassium transients. These results indicate that homeostatic mechanisms maintain spontaneous activity in the pre-hearing period, with significant implications for both circuit development and therapeutic approaches aimed at treating congenital forms of deafness arising through mutations in key sensory transduction components.

Human Plasma Rich in Growth Factors Improves Survival and Neurite Outgrowth of Spiral Ganglion Neurons In Vitro.

BACKGROUND: Platelet-rich and platelet-poor plasma (PRP and PPP) are autologous preparations from peripheral blood and contain several growth factors and cytokines involved in tissue repair. Although their neuroprotective and neuroregenerative properties have been already described, little is known about their effects in the inner ear. We, therefore, examined the effects of PRP and PPP on spiral ganglion neurons (SGN) in vitro. RESULTS: For all experiments, spiral ganglia were isolated from neonatal rats and were cultured in serum-free medium. PRP from human venous blood was added to dissociated SGN. Treatment with PRP (1:10, 1:50) significantly increased the neuronal survival and the neuronal outgrowth of SGN. This effect was completely reversed by the addition of Bay 11 (nuclear factor kappa B-inhibitor) and SB203580 (p38 mitogen-activated protein kinase [p38MAPK]-inhibitor). Furthermore, PPP was used as a cell-free matrix for the attachment of spiral ganglion explants. Coating with activated PPP improved the adhesion and neurite outgrowth of spiral ganglia explants. Therefore, activated PPP is a promising alternative for poly d/l-ornithine and laminin coating due to the gelatinous composition through the activation of PPP with calcium gluconate. PRP promotes neuroprotective and neuroregenerative effects on SGN when administered in adequate concentrations. These beneficial effects seem to be depending on NF-κB and the p38MAPK pathways. CONCLUSION: Preparations from autologous whole blood (PRP and PPP, respectively) present an interesting alternative for pharmacological intervention to the inner ear since they contain a balanced and natural composition of trophic factors.

The protective role of tetramethylpyrazine against cisplatin-induced ototoxicity.

OBJECTIVES: The aim of the present study was to investigate the protective effect of tetramethylpyrazine (TMP) on cisplatin-induced ototoxicity in rats. METHODS: Forty healthy, female, 24-week-old, Sprague-Dawley rats (n = 40) were randomly assigned to four groups as follows: group one (n = 10) received intraperitoneal (i.p.) physiological saline at daily doses of 3 mg/kg for seven days; group two (n = 10) received a single dose of i.p. 15 mg/kg cisplatin; group three (n = 10) received i.p. 140 mg/kg TMP daily for seven days plus a single dose of i.p. 15 mg/kg cisplatin on the fourth day; group four (n = 10) received i.p. 140 mg/kg TMP daily for seven days. Auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) measurements were obtained from the animals (40 rats, 80 ears) under general anesthesia before and after drug administration. The temporal bulla of animals were bilaterally removed for immunohistopathological examination. RESULTS: In group two, DPOAE and ABR values were significantly deteriorated after drug administration, whereas there was no statistically significant difference between the pre- and posttreatment DPOAE and ABR values for all frequencies for groups one, three and four. The mean scores for external ciliated cells (ECCs), stria vascularis (SV) and spiral ganglion (SG) injuries in hematoxylin and eosin (H&E) staining, and also caspase-3 immunoreactivity were significantly higher in group two than in the other groups. CONCLUSION: In the present study, the protective effect of TMP on cisplatin ototoxicity was demonstrated through studies of electrophysiology and immunohistopathology. Co-administration of TMP may have potential protective effects against cisplatin-induced ototoxicity.

Noise Stress Induces an Epidermal Growth Factor Receptor/Xeroderma Pigmentosum-A Response in the Auditory Nerve.

In response to toxic stressors, cancer cells defend themselves by mobilizing one or more epidermal growth factor receptor (EGFR) cascades that employ xeroderma pigmentosum-A (XPA) to repair damaged genes. Recent experiments discovered that neurons within the auditory nerve exhibit basal levels of EGFR+XPA co-expression. This finding implied that auditory neurons in particular or neurons in general have the capacity to mobilize an EGFR+XPA defense. Therefore, the current study tested the hypothesis that noise stress would alter the expression pattern of EGFR/XPA within the auditory nerve. Design-based stereology was used to quantify the proportion of neurons that expressed EGFR, XPA, and EGFR+XPA with and without noise stress. The results revealed an intricate neuronal response that is suggestive of alterations to both co-expression and individual expression of EGFR and XPA. In both the apical and middle cochlear coils, the noise stress depleted EGFR+XPA expression. Furthermore, there was a reduction in the proportion of neurons that expressed XPA-alone in the middle coils. However, the noise stress caused a significant increase in the proportion of neurons that expressed EGFR-alone in the middle coils. The basal cochlear coils failed to mobilize a significant response to the noise stress. These results suggest that EGFR and XPA might be part of the molecular defense repertoire of the auditory nerve.

Dopaminergic signaling in the cochlea: receptor expression patterns and deletion phenotypes.

Pharmacological studies suggest that dopamine release from lateral olivocochlear efferent neurons suppresses spontaneous and sound-evoked activity in cochlear nerve fibers and helps control noise-induced excitotoxicity; however, the literature on cochlear expression and localization of dopamine receptors is contradictory. To better characterize cochlear dopaminergic signaling, we studied receptor localization using immunohistochemistry or reverse transcriptase PCR and assessed histopathology, cochlear responses and olivocochlear function in mice with targeted deletion of each of the five receptor subtypes. In normal ears, D1, D2, and D5 receptors were detected in microdissected immature (postnatal days 10-13) spiral ganglion cells and outer hair cells but not inner hair cells. D4 was detected in spiral ganglion cells only. In whole cochlea samples from adults, transcripts for D1, D2, D4, and D5 were present, whereas D3 mRNA was never detected. D1 and D2 immunolabeling was localized to cochlear nerve fibers, near the first nodes of Ranvier (D2) and in the inner spiral bundle region (D1 and D2) where presynaptic olivocochlear terminals are found. No other receptor labeling was consistent. Cochlear function was normal in D3, D4, and D5 knock-outs. D1 and D2 knock-outs showed slight, but significant enhancement and suppression, respectively, of cochlear responses, both in the neural output [auditory brainstem response (ABR) wave 1] and in outer hair cell function [distortion product otoacoustic emissions (DPOAEs)]. Vulnerability to acoustic injury was significantly increased in D2, D4 and D5 lines: D1 could not be tested, and no differences were seen in D3 mutants, consistent with a lack of receptor expression. The increased vulnerability in D2 knock-outs was seen in DPOAEs, suggesting a role for dopamine in the outer hair cell area. In D4 and D5 knock-outs, the increased noise vulnerability was seen only in ABRs, consistent with a role for dopaminergic signaling in minimizing neural damage.

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.

Novel expression patterns of connexin 30.3 in adult rat cochlea.

Mutations of the GJB4 gene, encoding connexin 30.3 (CX30.3), are associated with skin disorders. Recently, this gene was also detected in deaf individuals without skin disorders. However, the functional roles of CX30.3 in the cochlea remain unclear. A primary step toward understanding the role of CX30.3 in hearing and its dysfunction is the documentation of its cellular and sub-cellular locations within the cochlea. In the present study, we located and determined the cellular expression of Cx30.3 within the rat cochlea by using a polyclonal anti-Cx30.3 antibody. Expression of the Cx30.3 protein was detected in the spiral limbus, spiral ligament, spiral ganglion, and stria vascularis by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) analyses. Our results indicate the presence and localization of Cx30.3 in the rat cochlea. Knowledge of the spatial distribution of Cx30.3 will provide important insights into its role in the cochleae and normal auditory function.

Expression of peripherin in the pig spiral ganglion--aspects of nerve injury and regeneration.

CONCLUSION: Peripherin protein may be important not only for developing neurons but also for the maintenance and regeneration of axonal processes in the mature cochlea. More knowledge about its expression and function could improve our understanding with reference to axonal regrowth and nerve restoration in the damaged cochlea. OBJECTIVE: To investigate the existence of peripherin protein in adult pig spiral ganglion and cultured spiral ganglion neurons of the guinea pig. MATERIALS AND METHODS: Immunohistochemistry with anti-peripherin antibodies was performed on sections of adult pig spiral ganglion and guinea pig spiral ganglion cell (SGC) culture. RESULTS: In pig auditory neurons, both type I and type II SGCs showed expression of the protein peripherin. These cells were not preferentially located near the intraganglionic spiral bundle (IGSB). The IGSB consisted of thin calibre fibres showing intense peripherin and thicker fibres that were TUJ-1 positive. Only a few fibres within the IGSB co-expressed both peripherin and TUJ-1. Cultured guinea pig neurons displayed a rich expression of peripherin, including the nuclei. This protein was expressed in regions such as the perikaryon and axons but there was also a segmental expression of peripherin in some regions. Peripherin was more expressed in areas of axon branching and in the centre of the active growth cones and lammelipodia.

Distribution of serotonin immunoreactivity in the spiral ganglion neurons of mouse cochlea.

OBJECTIVE: Serotonin (5-hydroxytryptamine, 5-HT) is a neuromodulator/neurotransmitter with multiple biological functions. Spiral ganglion cells in the cochlea are the primary neurons of the afferent system in the auditory transmission. In this study, we used the immunohistochemical technique to investigate the distribution of serotonin in the spiral ganglion of mouse cochlea. MATERIALS AND METHODS: The cochlea tissue of four adult mice was dissected and fixed. The immunohistochemical staining was applied by using goat anti-serotonin polyclonal antibody as primary antibody. Tissue sections were treated with biotin-labeled rabbit anti-goat immunoglobulin G, followed by adding streptavidin-biotin-peroxidase complex. Finally, the sections were stained with 3,3-diaminobenzidine (DAB) solution. RESULTS: The spiral ganglion exhibited pronounced immunoreactivity for serotonin. Specifically, serotonin immunoreactivity was detected in the cytoplasma of spiral ganglion neurons located in Rosenthal's canal of the bony modiolus of mouse cochlea. CONCLUSIONS: Since spiral ganglion neurons are the afferent neurons to the auditory sense organ, our result strongly suggests that serotonin molecule may function as a neuromodulator/neurotransmitter in the peripheral auditory processing.

Technical report: laser microdissection and pressure catapulting is superior to conventional manual dissection for isolating pure spiral ganglion fractions from the cochlea.

Isolating cells from the cochlea to perform molecular biology assessment presents a challenge, because it is not possible to dissect pure cell pools by conventional methods. Thus, we set out to demonstrate that laser microdissection and pressure catapulting (LMPC) is superior to conventional manual cochlea dissection for this purpose. Spiral ganglions (SG) were isolated from neonatal rat cochleae by manual dissection and LMPC. Also, modioli were manually dissected. Total RNA was isolated from all three cell pools. In order to demonstrate contamination of the dissected cell pool, we determined the expression of type II iodothyronine deiodinase (D2), claudin 11 (Cld-11), neurofilament light chain (NF-L) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts by RT-PCR. The results showed that LMPC is not only a suitable method for selectively dissecting cochlear tissues, but in addition the molecular markers confirmed pure spiral ganglion cell pools without indication for any contamination by other cells. This indicates that LMPC is capable of providing a pure SG cell pool in contrast to conventional manual dissection. Therefore, LMPC presents a new technique for cochlear tissue separation improving the validity of molecular biological studies of the inner ear.

Developmentally regulated expression of the P2X3 receptor in the mouse cochlea.

ATP-gated non-selective cation channels assembled from P2X(3) receptor subunits contribute to transduction and neurotransmitter signaling in peripheral sensory systems and also feature prominently in the development of the central nervous system. In this study, P2X(3) receptor expression was characterized in the mouse cochlea from embryonic day 18 (E18) using confocal immunofluorescence. From E18 to P6, spiral ganglion neuron cell bodies and peripheral neurites projecting to the inner and outer hair cells were labeled. The inner spiral plexus associated with the inner hair cell synapses had a stronger fluorescence signal than outer spiral bundle fibers which provide the afferent innervation to the outer hair cells. Labeling in the cell bodies and peripheral neurites diminished around P6, and was no longer detected after the onset of hearing (P11, P17, adult). In opposition to the axiom that P2X(3) expression is neuron-specific, inner and outer sensory hair cells were labeled in the base and mid turn region at E18, but at P3 only the outer hair cells in the most apical region of the cochlea continued to express the protein. These data suggest a role for P2X(3) receptor-mediated purinergic signaling in cochlear synaptic reorganization, and establishment of neurotransmission, which occurs just prior to the onset of hearing function.

Localization of neurotensin immunoreactivity in neurons and organ of Corti of rat cochlea.

The distribution of neurotensin-containing cell bodies and fibers has been observed in the central and peripheral nervous system, including sensory ganglia, but no description has been found in the peripheral auditory system. Here, we investigated the presence of neurotensin immunoreactivity in the cochlea of the adult Wistar rat. Strong neurotensin immunoreactivity was detected in the cytoplasm of the inner hair cells (IHC) and Deiters' cells of the organ of Corti. Outer hair cells (OHC) show weak immunoreaction. Neurotensin immunoreactivity was also found in the neurons and fibers of the spiral ganglia. Quantitative microdensitometric image analysis of the neurotensin immunoreactivity showed a strong immunoreaction in the hair cells of organ of Corti and a moderate to strong labeling in the spiral ganglion neurons. A series of double immunolabeling experiments demonstrated a strong neurotensin immunoreactivity in the parvalbumin immunoreactive IHC and also in the calbindin immunoreactive Deiters' cells. Weak neurotensin immunoreactivity was seen in the calbindin positive OHC. Neurofilament and parvalbumin immunoreactive neurons and fibers in the spiral ganglia showed neurotensin immunoreactivity. Calbindin immunoreactivity was not detected in the spiral ganglion neurons, which are labeled by neurotensin immunoreactivity. The presence of neurotensin in the cochlea may be related to its modulation of neurotransmission in the peripheral auditory pathway.

[Subunit of voltage-dependent calcium channels in the murine spiral ganglion cells].

OBJECTIVE: To investigate subunit type of voltage-dependent calcium channels in the spiral ganglion cells of the mouse. METHODS: The spiral ganglion cells were dissected from cochleae of neonatal mice and cultured for 24 h. Total RNA was extracted from cultured spiral ganglion cells. After reverse transcription, resulting cDNA was amplified by polymerase chain reaction (PCR) with primers targeted to nucleotide sequences corresponding to 7 different calcium channel subunits. The types of calcium channel subunits were identified by PCR analysis and nucleotide sequencing. RESULTS: Reverse transcription (RT)-PCR products representing subunit gene expression were strongly and consistently amplified for alpha1 D, alpha1 E, alpha2/delta, beta1 and beta3. Nucleotide sequencing confirmed the identity of mouse cochlear subunit cDNAs. CONCLUSIONS: alpha1D, alpha1E, alpha2/delta, beta1 and beta3 subunits are expressed in spiral ganglion cells. And the coexpression of alpha1D and alpha1 E demonstrate the presence of L-type and R-type calcium channels in mammalian spiral ganglion cells.

Synaptophysin immunohistochemistry in the human cochlea.

Light microscopy and immunohistochemical analyses of a freshly prepared human cochlea, removed at meningioma skull base surgery, were performed with particular emphasis on synaptophysin (SY) reactivity. Synaptophysin, a 38-kDa glycoprotein, is one of the most abundant integral membrane proteins of small presynaptic vesicles and is a useful marker for sites of synaptic transmission of the efferent olivocochlear system in the cochlea. Following fixation and decalcification, cryosections of 30 microm were prepared. To introduce immunostaining, free-floating sections were exposed to monoclonal SY antibody. Positive SY immunostaining was solely restricted to the neural and sensory structures and did not include supporting cells of the organ of Corti. Dense reaction products were noted around the hair cells, especially at the basal portion of the inner and outer hair cells and their neural poles, as well as around the inner spiral bundle, tunnel spiral bundle, outer spiral bundle and upper tunnel crossing fibers. The majority of spiral ganglion cells stained positively. An intermingling network of thin unmyelinated nerve fibers stained densely, especially at the basal portions of the cochlea. The spiral limbus, inner and outer sulcus cells, basilar membrane, myelinated nerve fibers, spiral ligament and the stria vascularis were unstained. Human cochlea obtained during surgery offers excellent conditions for immunohistochemical analysis. In the basal cochlea in the organ of Corti, outer hair cell area, there may be alterations due to noise trauma from the drilling procedure.

Immunolocalization of voltage-gated calcium channel alpha1 subunits in the chinchilla cochlea.

The immunohistochemical localization of alpha1A, alpha1B, alpha1C, alpha1D, and alpha1E voltage-gated calcium channel subunits was investigated in the chinchilla organ of Corti and spiral ganglia with the use of specific antipeptide antibodies. The inner and outer hair cells were immunoreactive for alpha1A and alpha1D subunit antibodies. Alpha1C immunoreactivity localized to the nerve terminals innervating inner hair cells and the basal pole of the outer hair cell. There was only non-specific staining to alpha1B and alpha1E. Supporting cells were non-immunoreactive. Spiral ganglia neurons were alpha1B, alpha1C, and alpha1D immunoreactive. A few spiral ganglia neurons were alpha1E immunoreactive. The importance of alpha1D, the pore-forming subunit of the L-type channel, in outer and inner hair cell function has been clearly demonstrated in electrophysiological, molecular biological, and knockout models. The presence of alpha1A, the pore-forming subunit of the P/Q type channels, has not previously been demonstrated in inner and outer hair cells, and its function in the cochlear hair cell is unknown.

Localization of glucocorticoid receptors in the murine inner ear.

We performed an immunohistochemical investigation of the distribution of glucocorticoid receptors (GRs) in the murine inner ear and found that GRs were expressed extensively, but with various degrees of immunoreactivity in different regions. We observed the strongest GR expression in the type III fibrocytes of the spiral ligament. Although the immunoreactivity of the cochlear hair cells and of the vestibular sensory epithelia was weak, the neighboring cochlear supporting cells and the subepithelial regions of the vestibular sensory epithelia were immunostained. Staining for GRs was also positive in the spiral ganglia and vestibular ganglia, as well as in the endolymphatic sac. The role of GRs in the inner ear is discussed.

Simultaneous detection of both nitric oxide and reactive oxygen species in guinea pig vestibular sensory cells.

Gentamicin-induced production of reactive oxygen species (ROS) and of nitric oxide (NO) in the vestibular end organs of the guinea pig was investigated by applying two new fluorescence indicators, 4,5-diaminofluorescein diacetate for direct detection of NO and dihydrotetramethylrosamine for ROS. The vestibular sensory cells produced both NO and ROS after exposure to gentamicin. A nonspecific inhibitor of NO synthase, L-NAME, inhibited the production of NO but did not appear to affect the production of ROS following exposure to gentamicin. In contrast, a radical scavenger, D-methionine, or the neurotrophin BDNF suppressed the production of ROS, in turn stimulating NO production. These findings could indicate that both NO and ROS play an important role in aminoglycoside ototoxicity.

Cholinergic innervation of the chick basilar papilla.

Antibodies directed against choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine (ACh) and a specific marker of cholinergic neurons, were used to label axons and nerve terminals of efferent fibers that innervate the chick basilar papilla (BP). Two morphologically distinct populations of cholinergic fibers were labeled and classified according to the region of the BP they innervated. The inferior efferent system was composed of thick fibers that coursed radially across the basilar membrane in small fascicles, gave off small branches that innervated short hair cells with large cup-like endings, and continued past the inferior edge of the BP to ramify extensively in the hyaline cell area. The superior efferent system was made up of a group of thin fibers that remained in the superior half of the epithelium and innervated tall hair cells with bouton endings. Both inferior and superior efferent fibers richly innervated the basal two thirds of the BP. However, the apical quarter of the chick BP was virtually devoid of efferent innervation except for a few fibers that gave off bouton endings around the peripheral edges. The distribution of ChAT-positive efferent endings appeared very similar to the population of efferent endings that labeled with synapsin antisera. Double labeling with ChAT and synapsin antibodies showed that the two markers colocalized in all nerve terminals that were identified in BP whole-mounts and frozen sections. These results strongly suggest that all of the efferent fibers that innervate the chick BP are cholinergic.

Distribution of members of the PSD-95 family of MAGUK proteins at the synaptic region of inner and outer hair cells of the guinea pig cochlea.

PDZ-domain containing proteins of the MAGUK (membrane-associated guanylate kinase) family target, anchor, and cluster receptors and channels to subcellular sites. Among the MAGUK proteins, the members of the PSD-95 family (MAGUKs: PSD-95, PSD-93, SAP-97, and SAP-102) target and anchor glutamate receptors to the synaptic terminals. Associations of glutamate receptors with MAGUKs have been described in the brain but not in the cochlea. In this study, RT-PCR, immunofluorescence microscopy, and immunoelectron microscopy were used to investigate the presence and distribution of MAGUK proteins in the organ of Corti. The presence of the mRNA for PSD-95, PSD-93, SAP-97, and SAP-102 in the organ of Corti was confirmed by RT-PCR. Immunocytochemistry using a "pan-MAGUK" antibody, which recognizes all four MAGUK proteins, and selective antibodies against these proteins revealed that all four MAGUKs are present within the base of inner hair cells while all except SAP-97 are found within the base of the outer hair cells. In addition, PSD-93 and PSD-95 are found in postsynaptic afferent terminals on inner hair cells, while postsynaptic afferent terminals on outer hair cells have PSD-93.