Vestibular Dark Research Articles

Localization of melatonin and its receptors (melatonin 1a and 1b receptors) in the mouse inner ear

Background: In the inner ear, evidence has been gathered indicating that melatonin plays important roles in inner ear physiology and pathophysiology. However, no attempt has been made previously to investigate the localization or expression of melatonin and its receptors in the whole inner ear.Aims/objectives: To analyze the presence of melatonin and its receptors in the normal mouse inner ear.Material and methods: C57BL6/J mice were used in this study. The localizations of melatonin, MT1a and MT1b in the inner ear, i.e. cochlea, vestibular end organs, vestibular ganglion and endolymphatic sac (ES), were studied by immunohistochemistry.Results: The organ of Corti, spiral ganglion, vestibular ganglion, vestibular sensory cells, vestibular dark and transitional cells, and ES epithelial cells showed an immunofluorescence reaction to melatonin, MT1a and MT1b.Conclusion and significance: The present findings show that melatonin and its receptors (MT1a and MT1b) are present in the inner ear, thus supporting the hypothesis that melatonin plays a physiological role in the inner ear.

Changes in the inner ear structures in cystic fibrosis patients

ObjectiveAlthough prolonged use of antibiotics is very common in cystic fibrosis (CF) patients, no studies have assessed the changes in both cochlear and peripheral vestibular systems in this population. MethodsWe used human temporal bones to analyze the density of vestibular dark, transitional, and hair cells in specimens from CF patients who were exposed to several types of antibiotics, as compared with specimens from an age-matched control group with no history of ear disease or antibiotic use. Additionally, we analyzed the changes in the elements of the cochlea (hair cells, spiral ganglion neurons, and the area of the stria vascularis). Data was gathered using differential interference contrast microscopy and light microscopy. ResultsIn the CF group, 83% of patients were exposed to some ototoxic drugs, such as aminoglycosides. As compared with the control group, the density of both type I and type II vestibular hair cells was significantly lower in all structures analyzed; the number of dark cells was significantly lower in the lateral and posterior semicircular canals. We noted a trend toward a lower number of both inner and outer cochlear hair cells at all turns of the cochlea. The number of spiral ganglion neurons in Rosenthal's canal at the apical turn of the cochlea was significantly lower; furthermore, the area of the stria vascularis at the apical turn of the cochlea was significantly smaller. ConclusionsDeterioration of cochlear and vestibular structures in CF patients might be related to their exposure to ototoxic antibiotics. Well-designed case-control studies are necessary to rule out the effect of CF itself.

Localization of sirtuins in the mouse inner ear

Conclusion: It is suggested that SIRT1 and 3, and probably SIRT4 and 5, play an important role in the neuroprotection of the inner ear. SIRT2 may be related to neuroprotection and myelin sheath formation, while SIRT6 seems to have a significant role in maintaining the energy balance by metabolic regulation. Objective: To analyze the expression of sirtuins (SIRT1–7) in the normal mouse inner ear. Methods: CBA/J mice were used for this study. The localization of SIRT1–7 in the inner ear, i.e. cochlea, vestibular end organs, and endolymphatic sac, was investigated using real-time PCR and immunohistochemistry. Results: We found high levels of mRNA of all seven sirtuins in the inner ear. In the immunohistochemical study, SIRT1–7 were abundant in many inner ear structures, i.e. stria vascularis, inner and outer hair cells, spiral ganglion cells, vestibular sensory and ganglion cells, vestibular dark and transitional cells, and the endolymphatic sac.

Localization of aquaporins 1, 2, and 3 and vasopressin type 2 receptor in the mouse inner ear

Conclusion: It is suggested that aquaporins (AQPs) 1, 2, and 3, and vasopressin type 2 receptors (V2Rs) in the fluid transporting cells, such as stria vascularis, vestibular dark and transitional cells, and endolymphatic sac epithelial cells, have an important role in fluid transport in the inner ear, while those in the sensory and ganglion cells may play a functional role in the sensory cell transduction system. Objective: To analyze expression of AQP1, AQP2, and AQP3 as well as V2Rs in the normal mouse inner ear. Methods: CBA/J mice were used in this study. Localization of AQP1, AQP2, AQP3, and V2Rs in the inner ear, i.e. cochlea, vestibular end organs, and endolymphatic sac, was investigated by immunohistochemistry. Results: The results show that AQP1, AQP2, AQP3, and V2Rs are abundantly distributed in many inner ear structures, i.e. stria vascularis, inner and outer hair cells, spiral ganglion cells, vestibular sensory and ganglion cells, vestibular dark and transitional cells, and the endolymphatic sac.

P2U purinergic receptor inhibits apical IsK/KvLQT1 channel via protein kinase C in vestibular dark cells.

Vestibular dark cells (VDC) are known to electrogenically secrete K+ via slowly activating K+ (IsK) channels, consisting of IsK regulatory and KvLQT1 channel subunits, and the associated short-circuit current (Isc) is inhibited by agonists of the apical P2U (P2Y2) receptor (J. Liu, K. Kozakura, and D. C. Marcus. Audit. Neurosci. 2: 331-340, 1995). Measurements of relative K+ flux (JK) with a self-referencing K(+)-selective probe demonstrated a decrease in JK after apical perfusion of 100 microM ATP. On-cell macropatch recordings from gerbil VDC showed a decrease of the IsK channel current (IIsK) by 83 +/- 7% during pipette perfusion of 10 microM ATP. The magnitude of the decrease of Isc by ATP was diminished in the presence of inhibitors of phospholipase C (PLC) and protein kinase C (PKC), U-73122 and GF109203X. Activation of PKC by phorbol 12-myristate 13-acetate (PMA, 20 nM) decreased IIsK by 79 +/- 3% in perforated-patch whole cell recordings, whereas the inactive analog, 4 alpha-PMA, had no effect. In contrast, elevation of cytosolic Ca2+ concentration by A-23187 increased the whole cell IIsK. The expression of the isk gene transcript was confirmed, and the serine responsible for the species-specific response to PKC was found to be present in the gerbil IsK sequence. These data provide evidence consistent with a direct effect of the PKC branch of the PLC pathway on the IsK channel of VDC in response to activation of the apical P2U receptor and predict that the secretion of endolymph in the human vestibular system may be controlled by PKC in the same way as in our animal model.

K +-induced stimulation of K + secretion involves activation of the I sK channel in vestibular dark cells

Vestibular dark cells in the inner ear secrete K + from perilymph containing 4 mM K + to endolymph containing 145 mM K +. Sensory transduction causes K + to flow from endolymph to perilymph, thus threatening the homeostasis of the perilymphatic K + concentration which is crucial for maintaining sensory transduction since the basolateral membranes of the sensory cells and adjacent neuronal elements need to be protected from K +-induced depolarization. The present study addresses the questions (1) whether increases in the perilymphatic K + concentration by as little as 1 mM are sufficient to stimulate KCl uptake across the basolateral membrane of vestibular dark cells, (2) whether K +-induced stimulation of KCl uptake causes stimulation of the I sK channel in the apical membrane, and (3) whether the rate of transepithelial K + secretion depends on the perilymphatic (basolateral) K + concentration when the apical side of the epithelium is bathed with a solution containing 145 mM K +, as in vivo. Uptake of KCl was monitored by measuring cell height as an indicator for cell volume. The current ( I IsK, conductance ( g IsK) and inactivation time constant ( τ IsK of the I sK channel as well as the apparent reversal potential of the apical membrane ( V r) were obtained with the cell-attached macro-patch technique. V r was corrected for the membrane voltage previously measured with microelectrodes. The rate of transepithelial K + secretion J K was obtained as equivalent short circuit current from measurements of the transepithelial voltage ( V t) and resistance ( R t) measured in the micro-Ussing chamber. Cell height of vestibular dark cells was 7.2 μm (average). Elevations of the extracellular K + concentration from 3.5 to 4.5 mM caused cell swelling with an initial rate of cell height change of 11 nm/s. With 3.6 mM K + in the pipette I IsK was outwardly directed and elevation of the extracellular K + concentration from 3.6 to 25 mM caused an increase of I IsK from 12 to 65 pA, g IsK from 152 to 950 pS and τ IsK from 278 to 583 ms as well as a hyperpolarization of V r from −50 to −60 mV. With 150 mM K + in the pipette I IsK was inwardly directed and the elevation of the extracellular K + concentration caused an increase of I IsK from −1 to −143 pA, g IsK from 141 to 1833 pS and τ ISK from 248 to 729 ms. V r remained within ± 10 mV from zero. J K was 4.8 nmol × cm −2 × s −1 when the both the apical side and the basolateral side of the epithelium were perfused with a solution containing 3.5 mM K +. Elevation of the basolateral K + concentration by 1 mM caused J K to increase by 1.1 nmol × cm −2 × s −1 or 23%. When the basolateral side of the epithelium was perfused with a solution containing 3.5 mM K + and the apical side with a solution containing 145 mM K +, as in vivo, J K was 0.8 nmol × cm −2 × s −1 and elevation of the basolateral K + concentration by 1 mM caused J K to increase by 0.8 nmol × cm −2 × s −1 or 100%. These data suggest that physiologically relevant increases in the perilymphatic K + concentration increase J K by increasing KCl uptake across the basolateral membrane and activation of K + release via the I sK channel in the apical membrane. Thus, the data demonstrate that vestibular dark cells adjust the rate of K + secretion into endolymph according to the perilymphatic K + concentration.

Vestibular dark cells contain the [formula omitted] exchanger NHE-1 in the basolateral membrane

Vestibular dark cells and strial marginal cells transport K + by similar mechanisms. We have shown that K + transport in vestibular dark cells is sensitive to the cytosolic pH (pH i) (Wangemann et al. (1995a): J. Membrane Biol. 147: 255–262). The present study addresses pharmacologically the questions whether vestibular dark cells and strial marginal cells from the gerbil contain a Na + H + exchanger (NHE) and in which membrane, apical or basolateral, NHE is located. Further, the study addresses the question which NHE subtype is present in vestibular dark cells. pH i was measured micro-fluorometrically with the pH-sensitive dye 2′,7′-bicarboxyethyl-5(6)carboxyfluorescein (BCECF), cell volume which is a measure of the net balance between ion influx and efflux was monitored as cell height (CH) and the equivalent short circuit current ( I sc ) which is a measure of transepithelial K + secretion was calculated from measurements of the transepithelial voltage ( V t ) and the transepithelial resistance ( R t ). Changes in pH i were induced by 20 or 40 m propionate. In the presence of propionate a transient acidification of pH, was observed in vestibular dark cells as well as a subsequent alkalinization to pH; values exceeding those under control conditions. The alkalinization of pH i in the presence of propionate was inhibited by the NHE blockes amiloride and EIPA. Propionate-induced swelling of vestibular dark cells was inhibited by amiloride. The NHE blocker amiloride caused in vestibular dark cells an acidification of pH; and a decrease in CH. Amiloride caused in both vestibular dark cells and strial marginal cells a transient stimulation of I sc when added to the basolateral side but not when added to the apical side. Similar effects on pH i were observed in vestibular dark cells with the amiloride analog ethyl-isopropyl-amiloride (EIPA) and similar effects on I sc were observed with EIPA and the NHE blocker HOE694 when applied to the basolateral side of vestibular dark cell epithelium. The ICS, for these basolateral effects of EIPA, HOE694 and amiloride on I sc . in vestibular dark cells were 2 × 10 −7 M, 8×10 −7 M and 4 × 10 −5 M. These observations suggest that vestibular dark cells and strial marginal cells contain NHE in their basolateral membrane, that K + transport in strial marginal cells is pH i sensitive similar to K + transport in vestibular dark cells and that NHE in vestibular dark cells consists of the subtype NHE-1.

Comparison of ion transport mechanisms between vestibular dark cells and strial marginal cells

Morphologic similarities between strial marginal cells and vestibular dark cells have long been recognized and it has long been accepted that both of these cell types are involved in the secretion of K + into endolymph. Functional similarities of these two epithelia, however, were considered unlikely as long as strial marginal cells were assumed to generate the endocochlear potential which has no equivalent in the vestibular labyrinth. The recently introduced concept that strial marginal cells transport K + but that the mechanism for the generation of the endocochlear potential is located in another cell type provided the basis to hypothesize that ion transport mechanisms and their regulation are similar in vestibular dark and strial marginal cells. The present review compiles evidence in support of this hypothesis.

Expression of α and β subunit isoforms of Na,K-ATPase in the mouse inner ear and changes with mutations at the W v or Sl d loci

Mice homozygous for mutations at the viable dominant spotting (W v) and Steel-dickie (Sl d) loci exhibit a similar phenotype which includes deafness. The auditory dysfunction derives from failure of the stria vascularis to develop normally and to generate a high positive endocochlear potential (EP). Because strial function is driven by Na,K-ATPase its expression was investigated in inner ears of W v W v and Sl d Sl d mice and their wild-type littermates by immunostaining with antisera against four of the enzyme's subunit isoforms. Wild-type mice from two different genetic backgrounds showed an identical distribution of subunit isoforms among inner ear transport cells. Several epithelial cell types coexpressed the α1 and β1 subunits. Vestibular dark cells showed no reactivity for β1 but expressed abundant β2, whereas, strial marginal cells stained strongly for both β isoforms. The only qualitative difference between mutant and wild-type mice was the absence of β1 subunit in marginal cells of the mutant's stria. However, it is unlikely that this difference accounts for failure of mutants to generate a high EP because the β1 subunit is not present in the stria vascularis of either rats or gerbils with normal EP values. Strong immunostaining for Na,K-ATPase in lateral wall fibrocytes of normal mice along with diminished immunoreactivity in the mutants supports the concept that these strategically located transport fibrocytes actively resorb K + leaked across Reissner's membrane into scala vestibuli or effluxed from hair cells and nerves into scala tympani. It is further speculated that the resorbed K + normally is siphoned down its concentration gradient into the intrastrial space through gap junctions between fibrocytes and strial basal and intermediate cells where it is recycled back to endolymph via marginal cells. Thus, failure of mutants to generate a positive EP could be explained by the absence of intermediate cells which may form the final link in the conduit for moving K + from perilymph to the intrastrial compartment.

Effect of gentamycin on vestibular dark cells and melanocytes: an ultrastructural and cytochemical study.

Vestibular dark cells areas in gentamycin (GM)-intoxicated guinea pigs were examined ultrastructurally and cytochemically. After GM treatment the dark cells represented cytoplasmic vacuoles, wide intercellular space, less osmiophilic cytoplasm and irregularly shaped basal portions. Cytochemically, Na+,K(+)-ATPase activity was demonstrated on the basolateral folded plasma membranes of the dark cells similarly in both GM-intoxicated and control guinea pigs. These findings revealed that the dark cells still possessed a function of regulating the ionic composition of the endolymph, although they showed various structural changes. On the other hand, the melanocytes extended their cytoplasmic processes into the basement membrane and the intercellular space of the dark cells. Melanosomes markedly increased in number dose-dependently by GM treatment. The significance and the function of the melanocytes in the dark-cell areas are discussed.

The endolymphatic sac and inner ear homeostasis. II: Effect of glycerol on the sensory end organs with or without colchicine pretreatment

The effects of glycerol and colchicine on the sensory end organs of the inner ear were investigated in mice. Glycerol alone induced a widening of the intercellular spaces lining vestibular dark and transitional cells as well as the marginal cells of the stria vascularis. This was noted within 30 min after the injection of glycerol and was normalized again within 4 h after the injection. Colchicine induced some morphological changes in the inner ear sensory cells, such as dissociation of Golgi complexes etc. These isolated glycerol or colchicine injections did not cause any signs of inner ear functional impairment. Treatment with glycerol following pretreatment with colchicine, however, induced marked inner ear dysfunction with impaired sense of balance and audition. The inner ear morphology revealed a combination of changes as compared with what was observed after isolated treatment with glycerol or colchicine i.e. edema of the stria vascularis, and vestibular dark and transitional cells as well as dissociation of Golgi complexes in the sensory cells. The cochlea showed moderate endolymphatic hydrops. These findings indicate that colchicine affects the inner ear fluid regulating mechanisms which may lead to severe functional derangement after additional glycerol treatment. It is conceivable that the present experiment may serve as a useful model for further studies on inner ear changes related to endolymphatic hydrops and Ménière's disease.