Adult Rat Cochlea Research Articles

Hair cell specific NTPDase6 immunolocalisation in vestibular end organs: Potential role of purinergic signaling in vestibular sensory transduction

A complex extracellular nucleotide signalling system acting on P2 receptors is involved in regulation of cochlear function in the mammalian inner ear. Ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) are ectonucleotidases that regulate P2 receptor signalling pathways in mammalian tissues by hydrolysing extracellular nucleotides to the respective nucleosides. All enzymes from the CD39/ENTPD family (NTPDase1-8) are expressed in the adult rat cochlea, but their expression and distribution in the vestibular end organ is unknown. This report demonstrates selective expression of NTPDase6 by rat vestibular hair cells. Hair cells transducing both angular acceleration (crista ampullaris) and static head position (maculae of the utricle and saccule) exhibited strong immunolabelling with a bias towards the sensory pole and in particular, the hair cell bundle. NTPDase6 is an intracellular enzyme that can be released in a soluble form from cell cultures and shows an enzymatic preference for nucleoside 5'-diphosphates, such as guanosine 5'-diphosphate (GDP) and uridine 5'-diphosphate (UDP). The main function of NTPDase6 may be the regulation of nucleotide levels in cellular organelles by regulating the conversion of nucleotides to nucleosides. NTPDase6 immunolocalisation in the vestibular end organ could be linked to the regulation of P2 receptor signalling and sensory transduction, including maintenance of vestibular hair bundles.

Expression and distribution of creatine transporter and creatine kinase (brain isoform) in developing and mature rat cochlear tissues

Physiological processes in the cochlea associated with sound transduction and maintenance of the unique electrochemical environment are metabolically demanding. Creatine maintains ATP homeostasis by providing high-energy phosphates for ATP regeneration which is catalyzed by creatine kinase (CK). Cellular uptake of creatine requires a specific high affinity sodium- and chloride-dependent creatine transporter (CRT). This study postulates that this CRT is developmentally regulated in the rat cochlea. CRT expression was measured by quantitative real-time RT-PCR and immunohistochemistry in the postnatal (P0-P14) and adult (P22-P56) rat cochlea. The maximum CRT expression was reached at the onset of hearing (P12), and this level was maintained through to adulthood. CRT immunoreactivity was strongest in the sensory inner hair cells, supporting cells and the spiral ganglion neurons. Cochlear distribution of the CK brain isoform (CKB) was also assessed by immunohistochemistry and compared with the distribution of CRT in the developing and adult cochlea. CKB was immunolocalized in the organ of Corti supporting cells, and the lateral wall tissues involved in K(+) cycling, including stria vascularis and spiral ligament fibrocytes. Similar to CRT, CKB reached peak expression after the onset of hearing. Differential spatial and temporal expression of CRT and CK in cochlear tissues during development may reflect differential requirements for creatine-phosphocreatine buffering to replenish ATP consumed during energy-dependent metabolic processes, especially around the period when the cochlea becomes responsive to airborne sound.

Distribution of NTPDase5 and NTPDase6 and the regulation of P2Y receptor signalling in the rat cochlea

Membrane-bound ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) in the inner ear regulate complex extracellular purinergic type-2 (P2) receptor signalling pathways through hydrolysis of extracellular nucleoside 5'-triphosphates and diphosphates. This study investigated the distribution of NTPDase5 and NTPDase6, two intracellular members of the E-NTPDase family, and linked this to regulation of P2 receptor signalling in the adult rat cochlea. These extracellular ectonucleotidases preferentially hydrolyse nucleoside 5'-diphosphates such as UDP and GDP. Expression of both enzymes at mRNA and protein level was detected in cochlear tissues and there was in vivo release of soluble NTPDase5 and 6 into cochlear fluids. Strong NTPDase5 immunostaining was found in the spiral ganglion neurones and supporting Deiters' cells of the organ of Corti, while NTPDase6 was confined to the inner hair cells. Upregulation of NTPDase5 after exposure to loud sound indicates a dynamic role for NTPDase5 in cochlear response to stress, whereas NTPDase6 may have more limited extracellular roles. Noise-induced upregulation of co-localised UDP-preferring P2Y(6) receptors in the spiral ganglion neurons further supports the involvement of NTPDase5 in regulation of P2Y receptor signalling. Noise stress also induced P2Y(14) (UDP- and UDP-glucose preferring) receptor expression in the root processes of the outer sulcus cells, but this was not associated with localization of the E-NTPDases.

Developmentally regulated expression of ectonucleotidases NTPDase5 and NTPDase6 and UDP-responsive P2Y receptors in the rat cochlea

Ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) regulate complex extracellular P2 receptor signalling pathways in mammalian tissues by hydrolysing extracellular nucleotides to the respective nucleosides. All enzymes from this family (NTPDase1-8) are expressed in the adult rat cochlea. This study reports the changes in expression of NTPDase5 and NTPDase6 in the developing rat cochlea. These two intracellular members of the E-NTPDase family can be released in a soluble form and show preference for nucleoside 5'-diphosphates, such as UDP and GDP. Here, we demonstrate differential spatial and temporal patterns for NTPDase5 and NTPDase6 expression during cochlear development, which are indicative of both cytosolic and extracellular action via pyrimidines. NTPDase5 is noted during the early postnatal period in developing sensory hair cells and supporting Deiters' cells of the organ of Corti, and primary auditory neurons located in the spiral ganglion. In contrast, NTPDase6 is confined to the embryonic and early postnatal hair cell bundles. NTPDase6 immunolocalisation in the developing cochlea underpins its putative role in hair cell bundle development, probably via cytosolic action, whilst NTPDase5 may have a broader extracellular role in the development of sensory and neural tissues in the rat cochlea. Both NTPDase5 and NTPDase6 colocalize with UDP-preferring P2Y(4), P2Y(6) and P2Y(14) receptors during cochlear development, but this strong association was lost in the adult cochlea. Spatiotemporal topographic expression of NTPDase5 and NTPDase6 and P2Y receptors in adult and developing cochlear tissues provide strong support for the role of pyrimidinergic signalling in cochlear development.

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 and localization of Tmie in adult rat cochlea

Loss-of function mutations in transmembrane inner ear expressed (Tmie/TMIE) gene have been shown to cause deafness in mice and humans (DFNB6). However, the functional roles of TMIE in the cochlea remain unclear. A primary step toward the understanding of the role of TMIE in hearing and its dysfunction is the documentation of its cellular and sub-cellular location within the cochlea, the auditory organ. In this study, we located and determined the cellular expression of Tmie within the rat cochlea using a polyclonal anti-Tmie antibody. The anti-Tmie antibody identified a specific band of 17 kDa in a variety of rat tissues by using Western blot analyses. The expression products of Tmie were also detected in the spiral limbus, spiral ligament, organ of Corti, and stria vascularis by immunohistochemistry analysis and RT-PCR. Our results point out the presence and localization of Tmie products in the cochlea of rat. Knowledge of spatial distribution of Tmie will provide important insight into the mechanisms that lead to deafness due to mutations in the TMIE gene.

FXYD6 Is a Novel Regulator of Na,K-ATPase Expressed in the Inner Ear

The exquisite sensitivity of the cochlea, which mediates the transduction of sound waves into nerve impulses, depends on the endolymph ionic composition and the endocochlear potential. A key protein in the maintenance of the electrochemical composition of the endolymph is the Na,K-ATPase. In this study, we have looked for the presence in the rat inner ear of members of the FXYD protein family, recently identified as tissue-specific modulators of Na,K-ATPase. Only FXYD6 is detected at the protein level. FXYD6 is expressed in various epithelial cells bordering the endolymph space and in the auditory neurons. FXYD6 co-localizes with Na,K-ATPase in the stria vascularis and can be co-immunoprecipitated with Na,K-ATPase. After expression in Xenopus oocytes, FXYD6 associates with Na,K-ATPase alpha1-beta1 and alpha1-beta2 isozymes, which are preferentially expressed in different regions of the inner ear and also with gastric and non-gastric H,K-ATPases. The apparent K(+) and Na(+) affinities of alpha1-beta1 and alpha1-beta2 isozymes are different. Association of FXYD6 with Na,K-ATPase alpha1-beta1 isozymes slightly decreases their apparent K(+) affinity and significantly decreases their apparent Na(+) affinity. On the other hand, association with alpha1-beta2 isozymes increases their apparent K(+) and Na(+) affinity. The effects of FXYD6 on the apparent Na(+) affinity of Na,K-ATPase and the voltage dependence of its K(+) effect are distinct from other FXYD proteins. In conclusion, this study defines the last FXYD protein of unknown function as a modulator of Na,K-ATPase. Among FXYD protein, FXYD6 is unique in its expression in the inner ear, suggesting a role in endolymph composition.

Differential expression of ryanodine receptors in the rat cochlea

This study examined the localization and functional expression of ryanodine receptors (RyR) within the cochlea using a combination of reverse transcription–polymerase chain reaction, immunolabeling techniques, and confocal Ca2+ imaging. All three RyR isoform mRNA transcripts were detected in the adult rat cochlea. Immunoperoxidase and immunofluorescence labeling showed that the three isoforms were differentially expressed. The most pronounced RyR protein expression, involving all three isoforms, occurred in the cell bodies of the spiral ganglion neurons. RyR3 labeling extended to the synaptic terminals innervating the inner and outer hair cells. RyR2 expression also occurred in the inner hair cells and supporting cells of the organ of Corti, while cells associated with ion homeostasis in the cochlea, such as the interdental cells of the spiral limbus (RyR1), and the epithelial cells of the spiral prominence and basal cells of the stria vascularis (RyR2 and RyR3), were also immunopositive. The functionality of RyR-gated Ca2+ stores in the spiral ganglion neurons was shown by confocal calcium imaging of fluo-4 fluorescence in rat cochlear slices. Caffeine (5mM) evoked an increase in intracellular Ca2+ concentration in the cell bodies of the spiral ganglion neurons which occurred inthe absence of external Ca2+. Ryanodine (50nm–1μM) evoked comparable increases in intracellular Ca2+ concentration. These findings suggest that RyR-mediated Ca2+ release may be involved in auditory neurotransmission, sound transduction, and cochlear electrochemical homeostasis.

Expression patterns of connexin 29 (GJE1) in mouse and rat cochlea

Multiple types of connexin ( Cxs) products, including Cx26, Cx30, Cx31, and Cx43, are found by immunolabeling in the mature cochlea. The transcript of Cx29, a newly discovered member of Cx gene family, was also discovered in the cochlea by cDNA macroarray hybridization. However, the functional roles of Cx29 in the cochlea remain unclear. To elucidate whether the Cx29 gap junction protein epsilon 1, GJE1, is localized in the adult mouse and rat cochlea, we performed an immunohistochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR) analysis. GJE1 was detected in the cochlea neurons, spiral limbus, spiral ligament, organ of Corti, and stria vascularis using IHC analysis. We also show that Cx29 mRNA is present in spiral limbus, spiral ligament, organ of Corti, stria vascularis, and lateral wall by the method of RT-PCR. Higher levels of Cx29 mRNA were found in spiral ligament and spiral limbus, whereas lower level in lateral wall. Our data first provide a comprehensive and detailed pattern of Cx29 gene expression in the mouse and rat cochlea. Knowledge of spatial distribution of Cx29 also allows the identification of candidate genes for deafness and provides important insight into mechanisms that lead to deafness due to mutations in Cx29 gene.

Expression of VASP and zyxin in cochlear pillar cells: indication for actin-based dynamics?

Vasodilator-stimulated phosphoprotein (VASP) is a member of the ENA/VASP-protein family. VASP is considered to be a crucial factor in the regulation of actin dynamics, which involves processes such as motility and cell adhesion, e.g. in filopodia or growth cones. In these processes zyxin acts as an important partner of VASP and is particularly concentrated at sites where VASP-dependent actin dynamics occur. Based on indirect evidence that actin-mediated dynamics may effect the mechanical properties of the cochlea, we have investigated expression of VASP and zyxin in the postnatal and adult rat cochlea using polymerase chain reaction and Western blot approaches, as well as immunohistochemistry and confocal microscopy. Besides an expected expression in vessels and fibroblasts, VASP and zyxin expression was also observed in pillar cells. Here, the staining was restricted to the head and foot plate of the pillar cells. Onset of VASP expression in pillar cells coincided with the beginning of hearing. In pillar cells, VASP and zyxin were co-localised with pan-actin, suggesting actin-based dynamics in these cochlear cells, which until now were rather presumed to form a highly rigid bridge between the inner and outer sensory cells. Thus, pillar cells may be more dynamically involved in controlling longer-lasting mechanical properties of the cochlea as hitherto presumed.

Expression of the P2X7 Receptor Subunit of the Adenosine 5’-Triphosphate-Gated Ion Channel in the Developing and Adult Rat Cochlea

ATP-gated ion channels assembled from P2X₇ subunits have been implicated in ontogeny and cellular pathology. Here, the expression of the P2X₇ receptor subunit was studied in the embryonic (E14–E18 days) and postnatal (P0–adult) rat cochlea using immunohistochemistry. Strong P2X₇ immunolabelling was observed in the primary auditory neurons of the spiral ganglion from E18 to adult and in the fibres innervating the sensory inner and outer hair cells from birth to adult. Strong immunolabelling of P2X₇ receptor protein was also observed in the inner and outer hair cells over a limited developmental period, from birth to P6. Weak expression was observed in cochlear duct epithelium on E18 and in the supporting cells (footplates of pillar cells in adult and in Böttcher’s cells after birth). The immunolocalisation of P2X₇ receptors further implicates extracellular ATP in signalling process in cochlear ontogeny and in establishment and function of auditory neurotransmission. The P2X₇ receptors may be involved in signal transduction and modulation as well as in regulating cell death during development and in pathological conditions.

Glial cell line-derived neurotrophic factor (GDNF) and its receptor complex are expressed in the auditory nerve of the mature rat cochlea

Glial cell line-derived neurotrophic factor (GDNF) is a survival factor for many neuronal cell types which signals through a heterodimer receptor consisting of GDNF-family receptor α 1 (GFRα-1) and Ret ( re arranged during t ransformation). GDNF expression has previously been reported in the inner hair cells of the rat cochlea, with expression of GFRα-1 but not Ret in the cell bodies of the auditory nerve (spiral ganglion cells), using in situ hybridization. The present study used reverse transcription-polymerase chain reaction (RT-PCR), and immunocytochemistry to examine GDNF, GFRα-1 and Ret in the adult rat auditory nerve. Semi-quantitative RT-PCR showed expression of GDNF and the two receptor components, GFRα-1 and Ret, in the modiolar subfraction of the cochlea containing spiral ganglion cells. A shorter mRNA splice variant for GDNF was also detected. Immunocytochemistry showed immunostaining in the modiolus for GDNF, GFRα-1 and Ret that was confined to spiral ganglion cells. When RT-PCR expression levels were compared to the expression in the substantia nigra, GFRα-1 expression levels were similar, Ret mRNA was lower in the modiolus and GDNF expression was higher in the modiolus. However, when GDNF was further assessed using Western blot, while GDNF protein was found in the modiolus it was at lower levels than in substantia nigra tissue. These results demonstrate that GDNF and both of its receptor components are found in spiral ganglion cells of the adult rat cochlea. Along with the previous report of GDNF in inner hair cells, these new results provide a basis for the role of GDNF as a survival factor for the auditory nerve, as suggested by previous studies.

Peripherin-like immunoreactivity in type II spiral ganglion cell body and projections

Peripherin, an intermediate filament protein, is present in neuronal subpopulations of both peripheral and central nervous systems. The distribution of peripherin was studied in the adult rat cochlea using immunohistochemistry on whole mount material, in cryostat sections and sections of plastic embedded tissue. In the spiral ganglion, peripherin labeling was restricted to the perikarya of a subpopulation of neurons and their peripheral and central processes. Peripherin positive neurons had the following features: (i) they have a large eccentric nucleus, they were often found in a cluster of 2 or 3 cells, (ii) they were often located near the intraganglionic spiral bundle fibers, (iii) they represented roughly 8% of the whole ganglion population and (iv) on the average they had smaller perikarya than non-immunoreactive cells. Immunostaining on semithin plastic sections revealed positive reactivity on Type II ganglion cells, while Type I neurons were negative. Double labeling using peripherin and three neurofilament (NF) subunit antibodies confirmed the presence of both markers within the same spiral ganglion cell type. Type II neurons have been previously documented as the only subpopulation of the spiral ganglion that presents a strong positive NF immunoreactivity within their perikarya. In the organ of Corti, peripherin-positive fibers formed bundles that course beneath the outer hair cells and send branches that end as boutons contacting the outer hair cells. All these characteristics suggest that peripherin-positive cells are Type II neurons, and that peripherin constitutes a reliable marker for this spiral ganglion subpopulation, as well as their peripheral and central processes.

Expression of mRNA encoding vasopressin V1a, vasopressin V2, and ANP-B receptors in the rat cochlea

The expression of mRNAs encoding vasopressin V1a, V2, and ANP-B receptors in the rat cochlea was examined by PCR and in situ hybridization. After reverse-transcription of rat cochlear RNA, cDNA was amplified by PCR using pairs of primers specific to these receptors. After subcloning of the PCR products, clones with sequences identical to those cloned previously from the rat liver (V1a receptor), kidney (V2 receptor) and brain (ANP-B receptor) were obtained. The localization of expression of those receptors in the developing and adult rat cochlea was examined by in situ hybridization using 35S-labeled cRNA probes. The V1a and V2 receptors were expressed throughout the whole of the neonatal rat cochlea, while no expression was detected in the adult cochlea. The ANP-B receptor was expressed throughout the whole of the neonatal cochlea. In the adult cochlea, expression was observed in the spiral ganglion and the spiral ligament. These results suggest that vasopressin may play a role in the development of the cochlea, and that natriuretic peptide may play a role in the function of the spiral ganglion and the spiral ligament.

Effect of superior cervical ganglionectomy on catecholamine concentration in rat cochlea

Both noradrenergic and dopaminergic nerve terminals have been described in the cochlea. The present report focused on the effect of superior cervical ganglionectomy (SCGx) on monoamine concentration in adult rat cochlea. In homogenates of whole cochleas, we measured the concentrations of norepinephrine (NE), dopamine (DA) and its main metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), by HPLC coupled to electrochemical detection. Measurements were carried out 4 h, 24 h or 6 days after unilateral SCGx. Most of the NE (∼82%) was lost after sympathectomy on the ipsilateral side, indicating that the principal localization of cochlear NE is in peripheral sympathetic fibers. Since about 18% of NE remained detectable 6 days after SCGx, a second origin of cochlear noradrenergic fibers may exist. Cochlear concentrations of DA or its metabolites did not change after SCGx. Therefore, DA and NE are located in two different populations of fibers within the cochlea, and are presumably related to distinct functional roles.

Histochemical localization of a β-galactoside-binding lectin and its binding-sites in developing and adult rat cochlea

The presence of an endogenous rat β-galactoside-specific lectin (S-Lac) and its specific binding ligands was analyzed, using a rabbit anti-human brain lectin of 114 kDa antibody (anti-HBL-14) and a probe consisting of a biotinylated derivative of the human lectin (HBL-biot) in adult and in developing rat cochleas. At PD1, some epithelial cells of the outer spiral sulcus (OSS) were specifically recognized by the anti-HBL-14 antibody. Then, anti-HBL-14 immunoreactivity progressively appeared in all OSS epithelial cells, and, from PD9 on, it also appeared in inner spiral sulcus (ISS) epithelial cells. Finally, young adult rats exhibited a well defined anti-HBL-14 immunoreactivity in epithelial cells of the inner and outer spiral sulcus. Using the biotinylated probe, from PD3 on, tectorial membrane (TM) and the sensory cells apical surfaces were labelled. The glycoconjugate expression within the developing organ of Corti seems to be highly complex. Lectin was detected in developing and adult epithelial cells surrounding the organ of Corti. In contrast, other glycoconjugates related to B and H human blood group antigens, were transiently present on sensory cells of the organ of Corti. Present findings can be correlated with significant events in development of the cochlea, mainly with cell-cell recognition or cellular adhesion.

Protein gene product 9.5 in the developing cochlea of the rat: cellular distribution and relation to the cochlear cytoskeleton.

Protein gene product 9.5 was immunolocalized in the adult and early postnatal (P2-P15) rat cochlea, and its distribution compared with a 200 kDa highly phosphorylated neurofilament subunit (neurofilament 200) and alpha-tubulin. In the adult, Protein gene product 9.5 was expressed exclusively in cochlear nerve fibres and ganglion cells, a small percentage of these (Type II ganglion cells and olivocochlear bundle fibres) being intensely positive for both protein gene product and neurofilament 200. In postnatal development, pillar and Deiters' cells were at first (P2-P15) strongly positive for protein gene product 9.5, and hair cells moderately so. At P2, all nerve fibres and ganglion cells showed co-expression of protein gene product 9.5 and neurofilament 200, but at later stages, the subset of intensely co-labelled neurons appeared, nerve fibres at P7 onwards and ganglion cells from P12. There was no overt correlation between the onset of protein gene product 9.5 and alpha-tubulin expression in any cochlear component. Protein gene product 9.5 expression in ganglion cells was at first (P2 and P7) mainly nuclear, and later also cytoplasmic. It is concluded that there is a clear correlation of high levels of protein gene product 9.5 and neurofilament protein expression, and that protein gene product 9.5 is expressed in some non-neuronal cells of the cochlea during its early development, persisting until after hearing has commenced.

HPLC detection of dopamine and noradrenaline in the cochlea of adult and developing rats

The presence and postnatal development of dopamine (DA) and noradrenaline (NA) in the rat cochlea were detected by high-performance liquid chromatography coupled with electrochemical detection. Cochlear DA content rose gradually after birth. Conversely, NA concentrations rose rapidly between postnatal day 1 and 8; then, up to day 30, it increased more slowly. On day 30, both DA and NA levels were around 5 times higher than on day 1. In the adult rat cochlea, NA mean content was 234 ± 22.2 pg/mg protein, while DA mean content was 23.6 ± 1.1 pg/mg protein. Adrenaline was always undetectable. The present study is the first report describing directly the presence of DA in the rat cochlea. DA might serve as one of the lateral efferent neurotransmitters, whereas NA probably acts as a neurotransmitter of the sympathetic cochlear innervation. Nevertheless, their influences on the cochlear physiology, either in adulthood or during development are still a matter of discussion.

Dose-dependent changes in the rat cochlea following aminoglycoside intoxication. II. Histological study.

The structural damages induced by a range of amikacin doses have been studied with SEM and TEM in the adult rat cochlea. Six groups of rats were given daily subcutaneous amikacin injections of either 200, 400, 600, 700, 800, or 1000 mg/kg per day for five consecutive days. Observations were made one month after the end of treatment, following functional recordings [(1987) Hear. Res. 26, 191-197]. In the group treated with 200 mg/kg per day, neither SEM nor TEM revealed any cochlear abnormalities. In the 400-mg group, the only damages were cytoplasmic abnormalities in the OHCs. Changes in the OHC stereocilia occurred at higher doses and were always associated with severe alterations of the underlying cell body. The first missing hair cells were OHCs in the apical regions of the cochleas of the 600-mg group. In the 700-mg group, the loss of OHCs extended to the middle and basal regions, mainly in the third row. By contrast, in the 800- and 1000-mg groups, the usual pattern of hair-cell degeneration was found. Loss of hair cells was greatest in the basal cochlear regions, where the first row of OHCs was more extensively destroyed. These results indicate that metabolic disorders (only visible with TEM) precede stereociliary alterations. Under SEM, the pattern of hair cell degeneration appeared to depend on the dose of the drug. The study clearly demonstrates that both SEM and TEM are needed to determine the true condition of hair cells after drug intoxication.