Histology and immunocytochemical localization of glial fibrillary acidic protein in the inner ear of Scincella tsinlingensis / Histología y localización inmunocitoquímica de la proteína ácida fibrilar glial en el oído interno de Scincella tsinlingensis

SUMMARY: The microstructure of inner ear in Scincella tsinlingensis was observed by light microscopy and the expression of glial fibrillary acidic protein (GFAP) in membranous labyrinth among the juvenile age group, subadult age group and adult age group were also detected by methods of immunohistochemistry. The inner ear in S. tsinlingensis resembled those in other Scincid lizards in their anatomy and histology. Large and elongate cochlear duct was slightly bowed or arched laterally. There was no hint of limbic modifications and the limbic lip was absent in cochlear recess. The basilar papilla elongated anteroventrally possessed specialized tectorial sallets. GFAP staining was significantly distributed in supporting cells of the sensory epithelia of cochlear duct, while the utricular macula and canal ampullae showed immunopositive for the GFAP antibody, with weaker staining in the saccular macula. The membranous inner ear of three different age groups revealed the similar pattern of GFAP expression, which suggested that the distribution of supporting cells were independent of age in S. tsinlingensis.

RESUMEN: La microestructura del oído interno en Scincella tsinlingensis fue analizada mediante microscopía óptica y por otra parte, fue cuantificada la expresión de la proteína ácida fibrilar glial (GFAP) en el laberinto membranoso, entre los grupos de edad juvenil, subadulto y adulto, utilizándose métodos inmunohistoquímicos. El oído interno de S. tsinlingensis se asemejaba al de otros lagartos Scincid tanto en su anatomía como en su histología. El conducto coclear mayor estaba ligeramente arqueado o arqueado lateralmente. No había indicios de modificaciones límbicas y no se evidenció el labio en el receso coclear. La papila basilar alargada anteroventralmente poseía sallets tectoriales especializados. La tinción de GFAP se distribuyó significativamente en las células del epitelio sensorial del conducto coclear, mientras que la mácula utricular y la ampolla del canal mostraron inmunopositividad para el anticuerpo GFAP, con una tinción más débil en la mácula sacular. El oído interno membranoso de los tres grupos de edad diferentes reveló un patrón similar de expresión de GFAP, lo que sugiere que la distribución de las células de soporte son independiente de la edad en S. tsinlingensis.

Arsenic level in toenails is associated with hearing loss in humans.

Arsenic (As) pollution in drinking water is a worldwide health risk for humans. We previously showed hearing loss in young people who live in areas of As-polluted drinking water and in young mice orally treated with As. In this study, we epidemiologically examined associations between As levels in toenails and hearing in 145 Bangladeshi aged 12-55 years in 2014. Levels of As in toenails, but not those in urine, were shown to be significantly correlated with hearing loss at 4 kHz [odds ratio (OR) = 4.27; 95% confidence interval (CI): 1.51, 12.05], 8 kHz (OR = 3.91; 95% CI: 1.47, 10.38) and 12 kHz (OR = 4.15; 95% CI: 1.55, 11.09) by multivariate analysis with adjustments for age, sex, smoking and BMI. Our experimental study further showed a significant association between As levels in inner ears and nails (r = 0.8113, p = 0.0014) in mice orally exposed to As, suggesting that As level in nails is a suitable index to assess As level in inner ears. Taken together, the results of our study suggest that As level in nails could be a convenient and non-invasive biomarker for As-mediated hearing loss in humans.

Spatiotemporal expression patterns of clusterin in the mouse inner ear.

Clusterin (CLU) is an extracellular chaperone protein that is implicated in diverse physiological and pathophysiological cellular processes. CLU expression is upregulated in response to cellular stress and under certain conditions, such as neurodegenerative disease and cancer. CLU primarily functions as a chaperone that exerts cytoprotective effects by removing cellular debris and misfolded proteins and also acts as a signaling molecule that regulates pro-survival pathways. Deafness is caused by genetic factors and various extrinsic insults, including ototoxic drugs, exposure to loud sounds and aging. Considering its cytoprotectivity, CLU may also mediate cellular defense mechanisms against hearing loss due to cellular stresses. To understand the function of CLU in the inner ear, we analyze CLU expression patterns in the mouse inner ear during development and in the adult stage. Results of quantitative real-time polymerase chain reaction analysis showed that Clu mRNA levels in the inner ear were increased during embryogenesis and were constantly expressed in the adult. Detailed spatial expression patterns of Clu both in the mRNA and protein levels were analyzed throughout various developmental stages via in situ hybridization and immunofluorescence staining. Clu expression was found in specific domains of developing inner ear starting from the otocyst stage, mainly adjacent to the prosensory domain of the cochlear epithelium. In the mature inner ear, Clu expression was observed in Deiter's cells and pillar cells of the organ of Corti, outer sulcus and in basal cells of the stria vascularis in the cochlea. These specific spatiotemporal expression patterns suggest the possible roles of CLU in inner ear development and in maintaining proper hearing function.

Innate immune defense in the inner ear - mucines are expressed by the human endolymphatic sac.

The human endolymphatic sac has been shown recently to have immunological capacities and has thus been proposed as the main entity protecting the inner ear from pathogen invasion, equivalent to mucosa-associated lymphoid tissue (MALT). Although the sac expresses molecules of the innate immune system, the potential expression of members of the important mucin family has not been detailed. Thus, this paper explores endolymphatic sac expression of a number of mucins and mucin precursors. Twelve fresh tissue samples from the human endolymphatic sac were obtained during translabyrinthine surgery. The expression of Mucin 1, 2, 5B/AC and 16, as well as the core structure elements (mucin precursors) T-antigen, Tn-antigen and Sialyl-Tn-antigen was investigated by immunohistochemistry. The endolymphatic sac epithelium expressed MUC1 (both apically towards the endolymphatic sac (ES) lumen and basally towards the capillary network), MUC 16 and Tn-antigen. There was no labeling after incubation with antibodies against T-antigen, sialyl-Tn-antigen, MUC2 and MUC5B/AC. We conclude that the human endolymphatic sac epithelium expresses a number of mucin molecules, which supports the hypothesis of the sac as the primary immunological tissue structure of the inner ear, equivalent to MALT in other organs. The mucins may also play a role in the formation and continuous homeostasis of the inner ear fluids, as well as the pathogenesis of Meniere's disease.

Altered expression of middle and inner ear cytokines in mouse otitis media.

OBJECTIVES/HYPOTHESIS: The inner ear is at risk for sensorineural hearing loss in both acute and chronic otitis media (OM), but the mechanisms underlying sensorineural hearing loss are unknown. Previous gene expression array studies have shown that cytokine genes might be upregulated in the cochleas of mice with acute and chronic OM. This finding implies that the inner ear could manifest a direct inflammatory response to OM that may cause sensorineural damage. Therefore, to better understand inner ear cytokine gene expression during OM, quantitative real-time polymerase chain reaction and immunohistochemistry were used in mouse models to evaluate middle and inner ear inflammatory and remodeling cytokines. STUDY DESIGN: Basic science experiment. METHODS: An acute OM model was created in Balb/c mice by a transtympanic injection of Streptococcus pneumoniae in one ear; the other ear was used as a control. C3H/HeJ mice were screened for unilateral chronic OM, with the noninfected ear serving as a control. RESULTS: Both acute and chronic OM caused both the middle ear and inner tissues in these two mouse models to overexpress numerous cytokine genes related to tissue remodeling (tumor necrosis factor-α, bone morphogenetic proteins, fibroblast growth factors) and angiogenesis (vascular endothelial growth factor), as well as inflammatory cell proliferation (interleukin [IL]-1α,ß, IL-2, IL-6). Immunohistochemistry confirmed that both the middle ear and inner ear tissues expressed these cytokines. CONCLUSIONS: Cochlear tissues are capable of expressing cytokine mRNA that contributes to the inflammation and remodeling that occur in association with middle ear disease. This provides a potential molecular basis for the transient and permanent sensorineural hearing loss often reported with acute and chronic OM.

Differences in gene expression between the otic capsule and other bones.

Our long term goal is to understand the molecular pathology of otosclerosis and to develop better forms of therapy. Toward this goal, the current study focused on characterizing the molecular factors responsible for the unique biological features of the otic capsule: its minimal rate of remodeling, and lack of healing capacity when fractured. We compared expression levels of 62 genes involved in bone metabolism between the adult murine otic capsule and the tibia and parietal bones; the latter exemplify bones formed by endochondral and intramembranous ossification, respectively. Gene expression levels were measured using real-time quantitative RT-PCR and analyzed using tools of bioinformatics. Expression patterns of key genes were verified with in situ hybridization. The molecular profile of the otic capsule was distinctly different from that of the tibia and parietal bone. Genes found to be most characteristic of the otic capsule were: osteoprotegerin (opg), bone morphogenetic protein receptor 1b (bmpr1b) and bone morphogenetic protein 3 (bmp3). Expression levels were high for opg and bmpr1b, and minimal for bmp3 within the otic capsule. We concluded that opg and bmpr1b likely play important roles in inhibition of remodeling within the otic capsule.

Modification of loop 1 affects the nucleotide binding properties of Myo1c, the adaptation motor in the inner ear.

Myo1c is one of eight members of the mammalian myosin I family of actin-associated molecular motors. In stereocilia of the hair cells in the inner ear, Myo1c presumably serves as the adaptation motor, which regulates the opening and closing of transduction channels. Although there is conservation of sequence and structure among all myosins in the N-terminal motor domain, which contains the nucleotide- and actin-binding sites, some differences include the length and composition of surface loops, including loop 1, which lies near the nucleotide-binding domain. To investigate the role of loop 1, we expressed in insect cells mutants of a truncated form of Myo1c, Myo1c(1IQ), as well as chimeras of Myo1c(1IQ) with the analogous loop from other myosins. We found that replacement of the charged residues in loop 1 with alanines or the whole loop with a series of alanines did not alter the ATPase activity, transient kinetics properties, or Ca(2+) sensitivity of Myo1c(1IQ). Substitution of loop 1 with that of the corresponding region from tonic smooth muscle myosin II (Myo1c(1IQ)-tonic) or replacement with a single glycine (Myo1c(1IQ)-G) accelerated the release of ADP from A.M 2-3-fold in Ca(2+), whereas substitution with loop 1 from phasic muscle myosin II (Myo1c(1IQ)-phasic) accelerated the release of ADP 35-fold. Motility assays with chimeras containing a single alpha-helix, or SAH, domain showed that Myo1c(SAH)-tonic translocated actin in vitro twice as fast as Myo1c(SAH)-WT and 3-fold faster than Myo1c(SAH)-G. The studies show that changes induced in Myo1c via modification of loop 1 showed no resemblance to the behavior of the loop donor myosins or to the changes previously observed with similar Myo1b chimeras.

Coordinated molecular control of otic capsule differentiation: functional role of Wnt5a signaling and opposition by sfrp3 activity.

Wnt proteins constitute one of the major families of secreted ligands that function in developmental signaling, however, little is known of the role of Wnt5a during inner ear development. It is hypothesized that Wnt5a acts as a mediator of chondrogenesis in the developing otic capsule, a cartilaginous structure that surrounds the developing inner ear and presages the formation of the endochondral bony labyrinth. We report the pattern of expression of Wnt5a protein and mRNA in the developing mouse inner ear using immunohistochemistry, whole-mount in situ hybridization and RT-PCR, and the ability of exogenous Wnt5a to stimulate otic capsule chondrogenesis when added to high-density cultures of periotic mesenchyme containing otic epithelium (periotic mesenchyme + otic epithelium), a well-established model of otic capsule formation. We show that in the presence of secreted frizzled related protein 3 (sfrp3), a Wnt antagonist expressed in the developing inner ear, or Wnt5a-specific antisense oligonucleotide, which diminishes endogenous Wnt5a, otic capsule chondrogenesis is suppressed in culture. We determined by histological analysis and aggrecan immunoreactivity that chondrogenic differentiation is disturbed in Wnt5a null embryos, and provide evidence that the periotic mesenchyme + otic epithelium harvested from Wnt5a null mice is compromised in its ability to differentiate into cartilage when interacted in culture. We propose a model whereby sfrp3 and Wnt5a act antagonistically to ensure appropriate patterns of chondrogenesis and provide coordinated control of otic capsule formation. Our findings support Wnt5a and sfrp3 as regulators of otic capsule formation in the developing mouse inner ear.

Calcium sensitivity of the cross-bridge cycle of Myo1c, the adaptation motor in the inner ear.

The class I myosin Myo1c is a mediator of adaptation of mechanoelectrical transduction in the stereocilia of the inner ear. Adaptation, which is strongly affected by Ca(2+), permits hair cells under prolonged stimuli to remain sensitive to new stimuli. Using a Myo1c fragment (motor domain and one IQ domain with associated calmodulin), with biochemical and kinetic properties similar to those of the native molecule, we have performed a thorough analysis of the biochemical cross-bridge cycle. We show that, although the steady-state ATPase activity shows little calcium sensitivity, individual molecular events of the cross-bridge cycle are calcium-sensitive. Of significance is a 7-fold inhibition of the ATP hydrolysis step and a 10-fold acceleration of ADP release in calcium. These changes result in an acceleration of detachment of the cross-bridge and a lengthening of the lifetime of the detached M-ATP state. These data support a model in which slipping adaptation, which reduces tip-link tension and allows the transduction channels to close after an excitatory stimulus, is mediated by Myo1c and modulated by the calcium transient.

Accumulation, fractionation, and analysis of platinum in toxicologically affected tissues after cisplatin, oxaliplatin, and carboplatin administration.

Antitumoral Pt-containing drugs present side effects like nephrotoxicity and ototoxicity. Several systematic experiments have been carried out with Wistar rats treated with cisplatin, carboplatin, and oxaliplatin to study Pt-drugs accumulation and elimination, and Pt-biomolecule distribution in the cells and cytosols of ear, kidney, and liver. Inductively coupled plasma-mass spectrometry (ICP-MS) analysis shows a cisplatin accumulation capability between oxaliplatin (the highest) and carboplatin (the lowest). The maximum concentration of Pt in all the organs studied was achieved around the first week after cisplatin treatment. During the first 30 days, the elimination was very fast, decreasing in the subsequent 60 days in all the organs. Analysis of cytosols by liquid chromatography (LC)-ICP-MS showed an analogous behavior. In most samples, the distribution of the three drugs in the cellular and cytosolic fractions was similar for all the tissues. For kidney and ear, approximately 60% and 30%, respectively, of the metal accumulated was present in the cytosol, the cytosolic fractions smaller than 50 KDa being especially important. Cisplatin-biomolecule interaction strength under denaturing conditions was evaluated by LC-ICP-MS and showed a quite strong bond.

Redox-dependent structural ambivalence of the cytoplasmic domain in the inner ear-specific cadherin 23 isoform.

Cadherin 23 (Cdh23), an essential factor in inner ear mechano-electric transduction, exists in two alternatively spliced forms, Cdh23(+68) and Cdh23(-68), depending on the presence and absence of exon 68. Cdh23(+68) is inner ear-specific. The exon 68-corresponding region confers an alpha-helical configuration upon the cytoplasmic domain (Cy) and includes a cysteine residue, Cys(3240). We demonstrate here that Cy(+68) as well as the transmembrane (TM) plus Cy(+68) region is present in two different forms in transfected cells, reduced and non-reduced, the latter existing in more compact configuration than the former. The observed characteristic of Cy(+68) was completely abolished by Cys(3240)Ala substitution. Treatment of TMCy(+68)-transfected cells with diethyl maleate, a glutathione depleting reagent, resulted in conversion of the non-reduced to the reduced form of TMCy(+68), suggesting glutathione to be a Cys(3240)-binding partner. Multiple alignment of mammalian Cdh23Cy sequences indicated the occurrence of conformation-inducible Cys in Cdh23Cy of mammals, but not lower vertebrates. The implications of Cys-dependent structural ambivalence of Cdh23 in inner ear mechanosensation are discussed.

Cochlin isoforms and their interaction with CTL2 (SLC44A2) in the inner ear.

Choline transporter-like protein 2 (CTL2) is a multi-transmembrane protein expressed on inner ear supporting cells that was discovered as a target of antibody-induced hearing loss. Its function is unknown. A 64 kDa band that consistently co-precipitates with CTL2 from inner ear extracts was identified by mass spectroscopy as cochlin. Cochlin is an abundant inner ear protein expressed as multiple isoforms. Its function is also unknown, but it is suspected to be an extracellular matrix component. Cochlin is mutated in individuals with DFNA9 hearing loss. To investigate the CTL2-cochlin interaction, antibodies were raised to a cochlin-specific peptide. The antibodies identify several cochlin polypeptides on western blots and are specific for cochlin. We show that the heterogeneity of the cochlin isoforms is caused, in part, by in vivo post-translational modification by N-glycosylation and, in part, caused by alternative splicing. We verified that antibody to CTL2 co-immunoprecipitates cochlin from the inner ear and antibody to cochlin co-immunoprecipitates CTL2. Using cochlear cross-sections, we show that CTL2 is more widely distributed than previously described, and its prominent expression on cells facing the scala media suggests a possible role in homeostasis. A prominent but previously unreported ribbon-like pattern of cochlin in the basilar membrane was demonstrated, suggesting an important role for cochlin in the structure of the basilar membrane. CTL2 and cochlin are expressed in close proximity in the inner sulcus, the spiral prominence, vessels, limbus, and spiral ligament. The possible functional significance of CTL2-cochlin interactions remains unknown.

Neurotrophin and Trk neurotrophin receptors in the inner ear of Salmo salar and Salmo trutta.

Neurotrophins (NTs) and their signal transducing Trk receptors play a critical role in the development and maintenance of specific neuronal populations in the nervous system of higher vertebrates. They are responsible for the innervation of the inner ear cochlear and vestibular sensory epithelia. Neurotrophins and Trks are also present in teleosts but their distribution in the inner ear is unknown. Thus, in the present study, we used Western-blot analysis and immunohistochemistry to investigate the expression and cell localization of both NTs and Trk receptors in the inner ear of alevins of Salmo salar and Salmo trutta. Western-blot analysis revealed the occurrence of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), but not nerve growth factor (NGF), as well as all three Trk receptors, i.e. TrkA, TrkB and TrkC, the estimated molecular weights of which were similar to those expected for mammals. Specific immunoreactivity for neurotrophins was detected mainly in the sensory epithelia. In particular, BDNF immunoreactivity was found in the maculae of the utricle and saccule, whereas NT-3 immunoreactivity was present in the sensory epithelium of the cristae ampullaris. As a rule the sensory epithelia of the inner ear lacked immunoreactivity for Trks, thus excluding possible mechanisms of autocrinia and/or paracrinia. By contrast, overlapping subpopulations of neurons in the statoacoustic ganglion expressed TrkA (about 15%), TrkB (about 65%) and TrkC (about 45%). The present results demonstrate that, as in mammals and birds, the inner ear of teleosts expresses the components of the neurotrophin-Trk system, but their roles remain to be elucidated.

Localization of anosmin-1a and anosmin-1b in the inner ear and neuromasts of zebrafish.

Anosmin-1, encoded by the KAL-1 gene, is the protein defective in the X-linked form of Kallmann syndrome. This human developmental disorder is characterized by defects in cell migration and axon target selection. Anosmin-1 is an extracellular matrix protein that plays a role, in vitro, in processes such as cell adhesion, neurite outgrowth, axon guidance, and axon branching. The zebrafish possesses two orthologues of the KAL-1 gene: kal1a and kal1b, which encode anosmin-1a and anosmin-1b, respectively. Previous in situ hybridization studies have shown that kal1a and kal1b mRNAs are expressed in undetermined cells of the inner ear but not in neuromast cells. Using specific antibodies against anosmin-1a and anosmin-1b, we report here that both proteins are expressed in sensory hair cells of the inner ear cristae ampullaris and the lateral line neuromasts. Accumulation of these proteins was observed mainly at the level of the hair bundle and also at the cell membrane. In neuromast hair cells, immunogold scanning electronmicroscopy demonstrated that anosmin-1a and anosmin-1b were present at the surface of the stereociliary bundle. In addition, anosmin-1a, but not anosmin-1b, was detected on the track of the ampullary nerve. This is the first report of anosmin-1 expression in sensory hair cells of the inner ear and lateral line, and along the ampullary nerve track.

Osteoprotegerin in the inner ear may inhibit bone remodeling in the otic capsule.

OBJECTIVES: To elucidate factors that may be responsible for the inhibition of remodeling of bone within the otic capsule. METHODS: Expression of osteoprotegerin (OPG), receptor activator of nuclear factor kappa B (RANK), and RANK ligand (RANKL) were assayed in samples of bone obtained from the otic capsule, calvarium, and femur, and from the soft tissue within the cochlea using semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) in mice. Immunostaining was used for histologic localization of the gene products. An enzyme-linked immunosorbent assay (ELISA) was used to quantify the amount of OPG within perilymph, serum, and cerebrospinal fluid. The micro-anatomy of the interface between the otic capsule and the fluid spaces of the cochlea was investigated by brightfield and phase-contrast microscopy and by three-dimensional reconstruction in the mouse and human. RESULTS: OPG, a powerful inhibitor of bone remodeling, was expressed at extremely high levels within the soft tissue of the cochlea and was present in the perilymph at very high concentrations. The OPG produced within the inner ear may diffuse into the surrounding otic capsule, where it may be responsible for inhibition of bone turnover. Our anatomic studies revealed an extensive system of interconnected canaliculi within the otic capsule that had direct openings into the fluid spaces of the inner ear, thus providing a possible anatomic route for the diffusion of OPG from the inner ear into the surrounding bone. CONCLUSION: OPG, a potent inhibitor of osteoclast formation and function, is expressed at high levels within the inner ear and is secreted into the perilymph and the surrounding bone and may serve to inhibit active bone remodeling within the otic capsule, especially immediately adjacent to the cochlea. By this means, the cochlear soft tissue may control the nature of the surrounding petrous bone.

Bone morphogenetic protein 4 (BMP4): a regulator of capsule chondrogenesis in the developing mouse inner ear.

Formation of the cartilaginous otic capsule is directed by otic epithelial-periotic mesenchymal interactions. In response to induction by otic epithelium, condensations of mesenchyme appear in the periotic region and form a chondrified otic capsule that serves as the template for the subsequent formation of the endochondral bony labyrinth. Previous studies indicate that members of the transforming growth factor beta superfamily, including transforming growth factor beta(1), participate in guiding these tissue interactions. In this study, we report the localization of bone morphogenetic protein 4 (BMP4) to the mesenchymal and epithelial-derived tissues of the mouse inner ear between 10.5 and 14 days of embryonic development. We demonstrate modulation of chondrogenesis in cultured mouse periotic mesenchyme by exogenous BMP4 protein and investigate the function of endogenous BMP4 in otic capsule chondrogenesis. We show that in the presence of the BMP antagonist, Noggin, otic capsule chondrogenesis is suppressed in culture in a dose-dependent manner. Consistent with this finding, addition of BMP4-specific antisense oligonucleotide to cultures of mouse periotic mesenchyme containing otic epithelium decreases levels of endogenous BMP4 protein and suppresses the chondrogenic response of the cultured periotic mesenchyme, providing evidence of the necessity for BMP4 in mediating otic capsule chondrogenesis. Supplementation of either Noggin- or BMP4 antisense oligonucleotide-treated cultures with BMP4 protein can restore the extent of chondrogenesis to normal levels. Our findings support BMP4 as an essential mediator of chondrogenesis in the developing otic capsule in situ.

Modulation of the voltage-gated sodium- and calcium-dependent potassium channels in rat vestibular and facial nuclei after unilateral labyrinthectomy and facial nerve transsection: an in situ hybridization study.

We investigated whether the expression in the vestibular and facial nuclei of the voltage-dependent Na alpha I and Na alpha III channels and of the Ca(2+)-activated K(+)-channel subunits, small-conductance (SK) 1, SK2 and SK3, is affected by unilateral inner-ear lesion including both labyrinthectomy and transsection of the facial nerve. Specific sodium (Na alpha I, Na alpha III) and potassium (SK1, SK2, SK3) radioactive oligonucleotides were used to probe sections of rat vestibular and facial nuclei by in situ hybridization methods. The signal was detected with films or by emulsion photography. Animals were killed at various times following the lesion: 1 day, 3 days, 8 days or 30 days. In normal adult animals, mRNAs for Na alpha I, and SK1, SK2, and SK3 channels were found in several brainstem regions including the lateral, medial, superior and inferior vestibular nuclei and the facial nuclei. In contrast, there was little Na alpha III subunit mRNA anywhere in the brainstem. Following unilateral inner ear lesion in rats, the medial vestibular nuclei were probed with Na alpha I, Na alpha III, SK1, SK2 and SK3 oligonucleotide probes: autoradiography indicated no difference between the two sides, at any of the times studied. Na alpha I and SK2 mRNAs were less abundant and Na alpha III, SK1 and SK3 mRNAs were more abundant in the axotomized facial nuclei motoneurons than in controls. Removal of vestibular input did not affect the abundance of the mRNAs for the sodium- or calcium-dependent potassium channels in the deafferented vestibular nuclei. There is thus no evidence that modulation of these conductances contributes to the recovery of a normal resting discharge of the deafferented vestibular neurons and consequently to the functional recovery of the postural and oculomotor deficits observed at the acute stage. However, facial axotomy induced a long-term modulation of both Na and SK conductances mRNAs in the facial motoneurons ipsilateral to the lesion. Presumably, retrograde injury factors resulting from axotomy were able to alter durably the membrane properties and thus the excitability of the facial motoneurons.

Classification and culture of spiral ligament fibrocytes from mice.

In this study, we established an immunocytochemical strategy to classify the fibrocytes of the murine spiral ligament (SL), and SL cultures were characterized. Similar to those in other mammals, three different types of fibrocytes were identified. Type I fibrocytes, which are found lateral to the stria vascularis, showed positive immunoreactivity for caldesmon and S-100 protein and were not stained for sodium-potassium-adenosinetriphosphatase (Na-K-ATPase). Type II fibrocytes are located lateral to the spiral prominence epithelium and suprastrial region, and they were distinguishable by their positive staining for Na-K-ATPase. Type III fibrocytes, which are found adjacent to bone in the inferior region of the SL, contained caldesmon but not S-100 or Na-K-ATPase. Secondary cultures from the SL were positive for caldesmon and S-100 and negative for Na-K-ATPase, suggesting that these cells were type I fibrocytes. The present immunocytochemical approach was useful for the classification of murine fibrocyte cultures, and these cultures may benefit future immunological studies of the inner ear because mice have been well characterized immunologically.

Role of osteopontin in the rodent inner ear as revealed by in situ hybridization.

Osteopontin (OPN) is considered to be a non-collagenous bone matrix protein that is involved in the ossification process. However, OPN has recently been observed in ectopic sites such as the kidney and nervous tissue. In the present study, the expression of OPN mRNA was examined in the rat and mouse inner ear by nonradioisotopic in situ hybridization. Signals of OPN mRNA were observed in the marginal cells of the stria vascularis, spiral ganglion cells, vestibular sensory hair cells, and vestibular dark cells. OPN protein was detected only in the otoconia by immunohistochemistry. The presence of OPN mRNA in the cochlea and vestibular dark cells may indicate that OPN is involved in the regulation of ions in the inner ear fluid. Findings in the saccule and utricle suggest that OPN is one of the protein components of the rodent otoconia and that the vestibular sensory hair cells are involved in the production of otoconia.