Deletion of Brg1 causes stereocilia bundle fusion and cuticular plate loss in vestibular hair cells.

Brg1 is an ATPase subunit of the SWI/SNF chromatin-remodeling complex, and it is indispensable for the development and homeostasis of various organs. Conditional deletion of Brg1 in cochlea hair cells (HCs) leads to multiple structural defects and profound deafness. However, the premature death of Brg1-deficient cochlea HCs hindered further study of the role of Brg1. In contrast to cochlea HCs, Brg1-deficient vestibular HCs survived for a long time. Therefore, HC apical structure and vestibular function were examined in inner HC-specific conditional Brg1 knockout mice. Vestibular HCs exhibited fused and elongated stereocilia bundles after deletion of Brg1, and the cuticular plate was absent in most HCs with fused stereocilia bundles. HC loss was observed in conditional Brg1 knockout mice at the age of 12 months. Morphological defects and HC loss were primarily restricted in the striolar region of the utricle and saccule and in the central region of ampulla. The behavioral tests revealed that Brg1 deletion in HCs caused vestibular dysfunction in older adult mice. These results suggest that Brg1 may play specific roles in the maintenance of the HC stereocilia bundle and the cuticular plate.

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.

Plasma membrane Ca2+-ATPase isoforms in frog crista ampullaris: identification of PMCA1 and PMCA2 specific splice variants.

Ca2+ ions play a pivotal role in inner ear hair cells as they are involved from the mechano-electrical transduction to the transmitter release. Most of the Ca2+ that enters into hair cells via mechano-transduction and voltage-gated channels is extruded by the plasma membrane Ca2+-ATPases (PMCAs) that operate in both apical and basal cellular compartments. Here, we determined the identity and distribution of PMCA isoforms in frog crista ampullaris: we showed that PMCA1, PMCA2 and PMCA3 are expressed, while PMCA4 appears to be negligible. We also identify PMCA1bx, PMCA2av and PMCA2bv as the major splice variants produced from PMCA1 and PMCA2 genes. PMCA2av appears to be the major Ca2+-pump operating at the apical pole of the cell, even if PMCA1b is also expressed in the stereocilia. PMCA1bx is, instead, the principal PMCA of hair cell basolateral compartment, where it is expressed together with PMCA2 (probably PMCA2bv) and PMCA3. Frog crista ampullaris hair cells lack a Na/Ca exchanger, therefore PMCAs are the only mechanism of Ca2+ extrusion. The coexpression of specific isozymes in the different cellular compartments responds to the need of a fine regulation of both basal and dynamic Ca2+ levels at the apical and basal pole of the cell.

Neuroprotection of vestibular sensory cells from gentamicin ototoxicity obtained using nitric oxide synthase inhibitors, reactive oxygen species scavengers, brain-derived neurotrophic factors and calpain inhibitors.

OBJECTIVE: In order to devise a new treatment for inner ear disorders, the efficacy of a nitric oxide synthase inhibitor (L-N(G)-nitroarginine methylester [L-NAME]), a radical scavenger (D-methionine), a neurotrophin (brain-derived neurotrophic factor [BDNF]) and a calpain inhibitor (leupeptin) for protection from hair cell damage was investigated. MATERIAL AND METHODS: The effects of these drugs on gentamicin-induced production of nitric oxide (NO) and reactive oxygen species (ROS) were studied by means of the fluorescence indicators 4,5-diaminofluorescein diacetate and dihydrotetramethylrosamine. The effect on gentamicin-induced vestibular hair cell damage was examined by using an in vitro LIVE/DEAD system. RESULTS: L-NAME inhibited the production of NO, D-methionine and BDNF restricted the production of ROS and leupeptin inhibited neither NO nor ROS. All the drugs used limited the vestibular hair cell damage caused by gentamicin. The combinations L-NAME + BDNF, L-NAME + leupeptin and D-methionine + BDNF had a significantly stronger preventive effect on hair cell damage. CONCLUSION: It is suggested that combined treatment with a radical inhibitor and either a neurotrophin or calpain inhibitor may help to treat inner ear disorders more effectively.

Blockade of c-Jun N-terminal kinase pathway attenuates gentamicin-induced cochlear and vestibular hair cell death.

The ototoxic action of aminoglycoside antibiotics leading to the loss of inner ear hair cells is well documented. However, the molecular mechanisms are poorly defined. We have previously shown that in neomycin-exposed cochlear organotypic cultures, the c-Jun N-terminal kinase (JNK) pathway - associated with stress, injury and apoptosis - is activated in hair cells. We have shown that hair cell death can be attenuated by CEP-1347, an inhibitor of JNK signaling (). In the present study, we demonstrate that gentamicin-induced ototoxicity leads to JNK activation and apoptosis in the inner ear hair cells in vivo. We show that systemic administration of CEP-1347 attenuates gentamicin-induced decrease of auditory sensitivity and cochlear hair cell damage. In addition, CEP-1347 treatment reduces the extent of hair cell loss in the ampullary cristae after gentamicin intoxication. Particularly, the inner hair cells of the cochlea and type I hair cells of the vestibular organs are protected. Our previous data have shown that also acoustic overstimulation can cause apoptotic death of cochlear hair cells and that CEP-1347 can attenuate noise-induced hair cell loss. Thus, our results imply that activation of JNK cascade may be a common molecular outcome of cellular stress in the inner ear sensory epithelia and that attenuation of the lesion can be provided by inhibiting JNK activation.

Major potassium conductance in type I hair cells from rat semicircular canals: characterization and modulation by nitric oxide.

Mammalian vestibular organs have two types of hair cell, type I and type II, which differ morphologically and electrophysiologically. Type I hair cells alone express an outwardly rectifying current, I(K, L), which activates at relatively negative voltages. We used whole cell and patch configurations to study I(K,L) in hair cells isolated from the sensory epithelia of rat semicircular canals. I(K,L) was potassium selective, blocked by 4-aminopyridine, and permeable to internal cesium. It activated with sigmoidal kinetics and was half-maximally activated at -74.5 +/- 1.6 mV (n = 35; range -91 to -50 mV). It was a very large conductance (91 +/- 8 nS at -37 mV; 35 nS/pF for a cell of average size). Patch recordings from type I cells revealed a candidate ion channel with a conductance of 20-30 pS. Because I(K,L) was activated at the resting potential, the cells had low input resistances (R(m)): median 25 MOmega at -67 mV versus 1.3 GOmega for type II cells. Consequently, injected currents comparable to large transduction currents (300 pA) evoked small (

Effects of L-arginine on the afferent resting activity in the cephalopod statocyst.

The effects of bath application of the nitric oxide (NO) precursor L-arginine (L-ARG) on the resting activity (RA) of afferent crista fibers were studied in isolated statocysts of the cuttlefish Sepia officinalis under various experimental conditions. L-ARG (threshold 10(-7) M) had three different effects: inhibition, excitation, and excitation followed by an inhibition; only the inhibitory effect of L-ARG was dose-dependent. D-Arginine (D-ARG) had no effect. When the preparation was pre-treated with NO synthase inhibitors (N(G)-Nitric-L-arginine methyl ester HCl (L-NAME), N(G)-Nitro-L-arginine (L-NOARG)), both the inhibitory and the excitatory effects of L-ARG significantly decreased at higher concentrations (10(-5 to -4) M), or were completely blocked at lower concentrations (10(-7 to -6) M), of L-ARG. When the preparation was pre-treated with guanylate cyclase inhibitors (1H-[1,2, 4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), methylene blue (M-BLU), cystamine (CYS)), L-ARG had only excitatory effects, whereas its effects were only inhibitory when the preparation was pre-treated with adenylate cyclase inhibitors 2',3'-dideoxyadenosine (DDA), MDL-12330A (MDL), nicotinic acid (NIC-A)). L-ARG had no effects when the pre-treatment was with a guanylate cyclase inhibitor and an adenylate cyclase inhibitor combined; in that situation, the RA of the afferent fibers remained. These data indicate that in cephalopod statocysts, a cGMP and a cAMP signal transduction pathway (presumably via the generation of NO) are responsible for the effects of L-ARG on the RA of crista afferent fibers. They also indicate that the L-ARG-cGMP pathway is the dominant pathway and is inhibitory, and that both pathways have only modulatory effects on, but are not essential for, the generation of the RA.

Involvement of nitric oxide in aminoglycoside vestibulotoxicity in guinea pigs.

Involvement of nitric oxide (NO) has been reported in physiological and pathological conditions in the inner ear. Recently, the presence of nitric oxide synthase (NOS) was demonstrated in the vestibular epithelium. In this study we used nicotinamide adenine dinucleotide phosphate-diapholase staining to monitor NOS activity during degeneration of guinea pig vestibular epithelia affected by streptomycin. Increased NOS activity was observed in affected epithelia in a dose- and time-dependent manner and a NOS inhibitor could protect hair cells from apoptosis. Additionally, cycloheximide significantly reduced NOS activity and the occurrence of apoptosis. These findings suggest that NO is involved in the degenerative process of vestibular epithelia caused by aminoglycosides.

Localisation of the nitric oxide (NO)/cGMP-pathway in the vestibular system of guinea pigs.

The exact distribution of nitric oxide-synthases (NOS) in the vestibular system has not been described satisfying yet. Immunostaining, using specific antibodies to the three known NOS-isoforms, to cyclic guanosine monophosphate (cGMP) and soluble guanylyl-cyclase (sGC), the second messenger system of nitric oxide (NO), was performed on paraffin sections of temporal bone from guinea pigs. eNOS could be detected in vestibular ganglion cells and in nerve fibres, including the calyces, surrounding the type 1 hair cells (HC). bNOS was found in the sensory epithelium, ganglion cells and in bone, while iNOS could not be found. NOS-detection was accompanied by reactivity to sGC and to cGMP. This finding implies that b- and eNOS-generated NO is involved in regulative processes in neurotransmission and regulation of blood flow.

Balance and hearing deficits in mice with a null mutation in the gene encoding plasma membrane Ca2+-ATPase isoform 2.

Plasma membrane Ca2+-ATPase isoform 2 (PMCA2) exhibits a highly restricted tissue distribution, suggesting that it serves more specialized physiological functions than some of the other isoforms. A unique role in hearing is indicated by the high levels of PMCA2 expression in cochlear outer hair cells and spiral ganglion cells. To analyze the physiological role of PMCA2 we used gene targeting to produce PMCA2-deficient mice. Breeding of heterozygous mice yielded live homozygous mutant offspring. PMCA2-null mice grow more slowly than heterozygous and wild-type mice and exhibit an unsteady gait and difficulties in maintaining balance. Histological analysis of the cerebellum and inner ear of mutant and wild-type mice revealed that null mutants had slightly increased numbers of Purkinje neurons (in which PMCA2 is highly expressed), a decreased thickness of the molecular layer, an absence of otoconia in the vestibular system, and a range of abnormalities of the organ of Corti. Analysis of auditory evoked brainstem responses revealed that homozygous mutants were deaf and that heterozygous mice had a significant hearing loss. These data demonstrate that PMCA2 is required for both balance and hearing and suggest that it may be a major source of the calcium used in the formation and maintenance of otoconia.

Localization of Ca-ATPase in frog crista ampullaris.

The distribution of Ca-ATPase in frog crista ampullaris was mapped ultracytochemically by using a one-step lead citrate reaction. Electron-dense precipitates, as an expression of Ca-ATPase activity, were observed on the surface of stereocilia and on the apical membrane surrounding the cuticular plate of hair cells. Sensory cells of the isthmus region showed more reactivity than those of the peripheral regions of the crista. No reaction products were detectable on the basolateral membranes and in cytoplasmatic organelles. Supporting cells of the crista showed a quite variable Ca-ATPase reaction on microvilli and on basolateral membranes. The presence of an evident reactivity on the stereocilia is consistent with the existence of an apical calcium microdomain involved in the mechano-transduction process and supports the current view that calcium ions enter the stereocilia during natural stimulation. On the other hand, the lack of an observable reactivity on the basolateral membrane of hair cells suggests that in semicircular canals other mechanisms of active transport of calcium ions across the plasma membrane, such as Na-Ca exchange, may be involved in homeostasis of the ion.

[Immunocytochemical study of cholinergic innervation in the neurosensory epithelia of human vestibule].

OBJECTIVE: To investigate the cholinergic innervation of the neurosensory epithelia of human vestibule. METHODS: A modified preembedding immunostaining technique for immunoelectronmicroscopy was applied to this study. A polyclonal antibody to choline acetyltransferase (ChAT) was used as the marker of cholinergic fibers. RESULTS: ChAT-immunoreactive products were restricted to the nerve fibers and terminals which were rich in synaptic vesicles. The ChAT-immunoreactive fibers synaps with afferent chalice as well as with type II sensory hair cells. CONCLUSION: This study demonstrates that cholinergic fibers innervate the neurosensory epithelia of human vestible. The cholinergic fibers of human vestibular sensory epithelia belong to the vestibular efferent system.

Acid phosphatase activity in the chick's inner ear.

The cellular localization of distribution of acid phosphatase (AP) in the plastic sections of newly hatched chick's inner ear were investigated utilizing an azo-coupling method. AP activity as evidenced by azo dye deposits were well defined and localized in various cells of membranous labyrinth. Intense AP activity was detectable in the supranuclear area of hair cells in the basilar papilla and vestibular sensory hair cells. As in the case of the other sites of AP activity, marked AP activity was seen in the supranuclear area of the transitional epithelia of crista ampullaris and in the supranuclear area, or diffusely in the cytoplasm of the dark cells at the base of crista ampullaris. The columnar cells and the cells of tegmentum vasculosum showed moderate to strong AP activity. The statoacoustic and vestibular ganglion cells showed various degrees of AP activity. On the AP activity of statoacoustic or vestibular ganglion, in comparison between the sections from JB-4 Plus embedded specimens and those from LR White embedded specimen, the latter could more intensely demonstrate AP activity than the former. Moreover, sections fixed in 2.5% paraformaldehyde demonstrated more intense AP activity than those fixed in 2.5% glutaraldehyde.

Development of vestibular and auditory function: effects of hypothyroidism and thyroxine replacement therapy on nystagmus and auditory evoked potentials in the pigmented rat.

The functional development of semicircular canals and some brainstem structures of the auditory system was followed in parallel with time in control and propylthiouracyl-induced hypothyroid pigmented rats by respective recording of postrotatory nystagmus response and auditory evoked brainstem potentials, with the aim of discovering the timing of permanent alterations of these responses in congenital hypothyroidism. A group of hypothyroid rats which under went thyroxine-replacement therapy from postnatal day 12 onward was also included in our studies to corroborate the involvement of thyroid hormones in these effects. Postrotatory nystagmus and auditory evoked responses were absent in congenital hypothyroid rats. In the thyroxine-replaced group postrotatory nystagmus values showed no differences from the control group from postnatal day 28 onward. Auditory evoked potentials in thyroxine-replaced animals could not be elicited at 30 dB, but by increasing the intensity of stimulus to 70 dB, values of latencies of the four waves composing the response were indistinguishable from controls from postnatal day 39 and thereafter. These results show that hypothyroidism affects both semicircular canal and auditory function, the latter more severely than the former, but that these effects can be prevented when thyroxine replacement treatment is started in early stages of postnatal development.

Histochemistry and role of nitric oxide synthase in the amphibian (Ambystoma tigrinum) inner ear.

Nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d) histochemistry was investigated in the axolotl (Ambystoma tigrinum) inner ear. Hair cells showed an intense NADPH-d reaction; afferent neurones also stained but less intensely than hair cells. Effects of NG-nitro-L-arginine (L-NOARG) on the basal discharge and mechanical responses of semicircular canal afferent neurones recorded extracellularly were also studied. L-NOARG (1 mu M) diminished the basal discharge and the response of afferent neurones to sinusoidal mechanical stimuli to 45 +/- 6.4% and 65 +/- 5.3% (mean +/- SEM) of control value, respectively. These findings suggest that production of nitric oxide (NO) by hair cells and probably also by afferent neurones contributes to the basal discharge and the response of afferent neurones to mechanical stimuli.