Mutations in a plasma membrane Ca2+-ATPase gene cause deafness in deafwaddler mice.

Hearing loss is the most common sensory deficit in humans. Because the auditory systems of mice and humans are conserved, studies on mouse models have predicted several human deafness genes and identified new genes involved in hearing. The deafwaddler (dfw) mouse mutant is deaf and displays vestibular/motor imbalance. Here we report that the gene encoding a plasma membrane Ca2+-ATPase type 2 pump (Atp2b2, also known as Pmca2) is mutated in dfw. An A-->G nucleotide transition in dfw DNA causes a glycine-to-serine substitution at a highly conserved amino-acid position, whereas in a second allele, dfw2J, a 2-base-pair deletion causes a frameshift that predicts a truncated protein. In the cochlea, the protein Atp2b2 is localized to stereocilia and the basolateral wall of hair cells in wild-type mice, but is not detected in dfw2J mice. This indicates that mutation of Atp2b2 may cause deafness and imbalance by affecting sensory transduction in stereocilia as well as neurotransmitter release from the basolateral membrane. These mutations affecting Atp2b2 in dfw and dfw2J are the first to be found in a mammalian plasma membrane calcium pump and define a new class of deafness genes that directly affect hair-cell physiology.

Analysis of miR-376 RNA cluster members in the mouse inner ear.

Mutations in phosphoribosyl pyrophosphate synthetase 1 (PRPS1) are associated with a spectrum of non-syndromic to syndromic hearing loss. PRPS1 transcript levels have been shown to be regulated by the microRNA-376 genes. The long primary RNA transcript of the miR-376 RNA cluster members undergo extensive and simultaneous A → I editing at one or both of two specific sites (+4 and +44) in particular human and mouse tissues. The PRPS1 gene, which contains target sites for the edited version of miR-376a-5p within its 3'UTR, has been shown to be repressed in a tissue-specific manner. To investigate whether the transcription of Prps1 is regulated by miR-376 cluster members in the mouse inner ear, we first quantified the expression of the mature miR-376 RNAs by quantitative real-time-PCR. The spatio-temporal patterns of miR-376 expression were assessed by in situ hybridization. Finally, we examined whether A →I editing of pri-miR-376 RNAs occurs in mouse inner ear by direct sequencing. Our data showed that the miR-376a-3p, b-3p, c-3p are present in mouse embryonic inner ears and intensive expression of miR-376a-3p/b-3p was detected in the sensory epithelia and ganglia of both auditory and vestibular portions of the inner ear. In adult inner ear, the expression of miR-376a-3p/b-3p is restricted within ganglion neurons of auditory and vestibular systems as well as the cells in the stria vascularis. Only unedited pri-miR-376 RNAs were detected in the cochlea suggesting that the activity of PRPS1 in the inner ear may not be regulated through the editing of miR-376 cluster.

Inner ear dysfunction in caspase-3 deficient mice.

BACKGROUND: Caspase-3 is one of the most downstream enzymes activated in the apoptotic pathway. In caspase-3 deficient mice, loss of cochlear hair cells and spiral ganglion cells coincide closely with hearing loss. In contrast with the auditory system, details of the vestibular phenotype have not been characterized. Here we report the vestibular phenotype and inner ear anatomy in the caspase-3 deficient (Casp3(-/-)) mouse strain. RESULTS: Average ABR thresholds of Casp3(-/-) mice were significantly elevated (P < 0.05) compared to Casp3(+/-) mice and Casp3(+/+) mice at 3 months of age. In DPOAE testing, distortion product 2F1-F2 was significantly decreased (P < 0.05) in Casp3(-/-) mice, whereas Casp3(+/-) and Casp3(+/+) mice showed normal and comparable values to each other. Casp3(-/-) mice were hyperactive and exhibited circling behavior when excited. In lateral canal VOR testing, Casp3(-/-) mice had minimal response to any of the stimuli tested, whereas Casp3(+/-) mice had an intermediate response compared to Casp3(+/+) mice. Inner ear anatomical and histological analysis revealed gross hypomorphism of the vestibular organs, in which the main site was the anterior semicircular canal. Hair cell numbers in the anterior- and lateral crista, and utricle were significantly smaller in Casp3(-/-) mice whereas the Casp3(+/-) and Casp3(+/+) mice had normal hair cell numbers. CONCLUSIONS: These results indicate that caspase-3 is essential for correct functioning of the cochlea as well as normal development and function of the vestibule.

Efferent neurotransmitters in the human cochlea and vestibule.

CONCLUSION: Current neurotransmission models based on animal studies on the mammalian inner ear not always reflect the situation in human. Rodents and primates show significant differences in characteristics of efferent innervation as well as the distribution of neuroactive substances. OBJECTIVE: Immunohistochemistry demonstrates the mammalian efferent system as neurochemically complex and diverse: several neuroactive substances may co-exist within the same efferent terminal. Using light and electron microscopic immunohistochemistry, this study presents a comparative overview of the distribution patterns of choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme, GABA, CGRP, and enkephalins within the peripheral nerve fiber systems of the human inner ear. MATERIALS AND METHODS: Human temporal bones were obtained post mortem and prepared according to a pre-embedding immunohistochemical technique to detect immunoreactivities to ChAT, GABA, CGRP, leu- and met-enkephalins at the electron microscopic level. RESULTS: Immunoreactivities of all the antigens were present within both the lateral and medial efferent systems of the cochlea, whereas only ChAT, GABA, and CGRP were detected in efferent pathways of the vestibular end organs.

Gastric-type H+,K+-ATPase in mouse vestibular end organs.

CONCLUSION: Gastric type H+,K+-ATPase in the vestibular end organs may be of importance for K+ circulation and may also be related to pH regulation in vestibular end organs and endolymphatic sac. OBJECTIVE: To analyze the expression of gastric-type H+,K+-ATPase in normal mouse vestibular end organs. METHODS: 8 weeks old CBA/J mice were used in this study. The presence of gastric-type H+,K+-ATPase α and ß in the vestibular end organs, viz. utricle, saccule, ampulla, vestibular ganglion, and endolymphatic sac, was investigated using immunohistochemistry. RESULTS: In the vestibular end organs, H+,K+-ATPase α and ß were almost identical. H+,K+-ATPase was expressed in sensory cells, the basolateral surface of dark cells, fibrocytes, in vestibular ganglion cells, and in the upper region of the endolymphatic sac epithelial cells.

Specific expression of the retinoic acid-synthesizing enzyme RALDH2 during mouse inner ear development.

Retinoid binding proteins and nuclear receptors are expressed in the developing mouse inner ear. Here, we report that the retinaldehyde dehydrogenase 2 (Raldh2) gene, whose product is involved in the enzymatic generation of retinoic acid (RA), exhibits a restricted expression pattern during mouse inner ear ontogenesis. The Raldh2 gene is first expressed at embryonic day (E) 10.5 in a V-shaped medio-dorsal region of the otocyst outer epithelium, which evolves as two separate domains upon otocyst morphogenesis. At E14.5, Raldh2 is expressed in two areas of the utricle epithelium and specific regions of the saccule and cochlear mesenchyme. Later, Raldh2 transcripts are restricted to two cochlear areas, the stria vascularis and Reissner membrane. Raldh2 mesenchymal expression did not correlate with migrating neural crest-derived melanoblasts. These restricted expression domains may correspond to specific sites of RA synthesis during inner ear morphogenesis.

Expression of the RSK2 gene during early human development.

The 90 kDa ribosomal S6 serine/threonine kinase 2 gene (RSK2, U08316) has been recently identified as a disease-causing gene in an X-linked disorder, the Coffin-Lowry Syndrome (MIM 303600) characterized by severe mental retardation, facial dysmorphisms and progressive skeletal malformations. To investigate its possible role in cerebral cortex development, we performed RNA in situ hybridization at three stages of human development: day 32 (Carnegie 15), 9 weeks (Carnegie 23) and 13 weeks. RSK2 expression is detected in the embryonic anterior and posterior telencephalon (hippocampus anlagen), mesencephalon, rhombencephalon and cerebellum. RSK2 gene expression is also observed in dorsal root ganglia, cranial nerve ganglia, and sensory epithelium of the inner ear, liver, lung and jaw anlagen. This pattern of expression may be involved in cognitive impairment and facial dysmorphisms found in Coffin-Lowry Syndrome.

Monoclonal L-citrulline immunostaining reveals nitric oxide-producing vestibular neurons.

Nitric oxide is an unstable free radical that serves as a novel messenger molecule in the central nervous system (CNS). In order to understand the interplay between classic and novel chemical communication systems in vestibular pathways, the staining obtained using a monoclonal antibody directed against L-citrulline was compared with the labeling observed using more traditional markers for the presence of nitric oxide. Brainstem tissue from adult rats was processed for immunocytochemistry employing a monoclonal antibody directed against L-citrulline, a polyclonal antiserum against neuronal nitric oxide synthase, and/or NADPH-diaphorase histochemistry. Our findings demonstrate that L-citrulline can be fixed in situ by vascular perfusion, and can be visualized in fixed CNS tissue sections by immunocytochemistry. Further, the same vestibular regions and cell types are labeled by NADPH-diaphorase histochemistry, by the neuronal nitric oxide synthase antiserum, and by our anti-L-citrulline antibody. Clusters of L-citrulline-immunoreactive neurons are present in subregions of the vestibular nuclei, including the caudal portion of the inferior vestibular nucleus, the magnocellular portion of the medial vestibular nucleus, and the large cells in the ventral tier of the lateral vestibular nucleus. NADPH-diaphorase histochemical staining of these neurons clearly demonstrated their multipolar, fusiform and globular somata and long varicose dendritic processes. These results provide support for the suggestion that nitric oxide serves key roles in both vestibulo-autonomic and vestibulo-spinal pathways.

Vestibular site of action of hypothyroidism in the pigmented rat.

The vestibular cell type affected by congenital hypothyroidism (CH) was investigated by measuring the activity of glutamate decarboxylase (GAD) and choline acetyltransferase (ChAT), synthesizing enzymes of putative afferent (GABA) and efferent (acetylcholine, ACh) neurotransmitters and thus, respectively, hair cell I and II (HC-I, HC-II), and efferent terminal (ET) marker enzymes, in vestibular homogenates of control, congenitally hypothyroid rats (CHR) and in thyroxine-replaced CHR (CHR-T4) whose postnatal age ranged from 20 to 60 days old. In the vestibule, CH-II and its efferent cholinergic contacting bouton mature prior to thyroid function whereas HC-I-differentiation and its efferent synapse arrival are the latest events in vestibular maturation. Therefore, a differential effect of CH upon GAD and ChAT in CHR could be anticipated. In control rats as in CHR the magnitude of GAD was the same with time starting on the 20th day. In CHR, ChAT gradually diminished beginning on day 28 to become 45% decreased with respect to control on the 60th postnatal day. Prevention of ChAT decrease in CHR by early administration of thyroxine (T4), a striking diminution of T4 and triiodothyronine (T3) in CHR serum and a normal level of these hormones found in CHR-T4 corroborated thyroid involvement. These results confirm the preference of hypothyroidism to affect cholinergic cell types (or compartments) of late maturation (HC-I-containing ET and hence 45% ChAT decrease) leaving HC-I, HC-II and HC-II-connecting ET untouched, supported by a 55% remanent ChAT and a constant GAD activity regardless of time and treatment.

Nitric oxide synthase in the vestibulocochlear system of mice.

The exact distribution of nitric oxide-synthases (NOS) and the NO-target enzyme soluble guanylyl cyclase (sGC) in the cochlea and vestibular organ is an issue of current discussion. The existence of NOS-isoforms in the cochlea of the guinea pig has been described recently, while information about the vestibular system are still rare and non-satisfying. In order to gain more information, immunostaining was performed, using specific antibodies to NOS I-III and to sGC, on paraffin sections of complete temporal bones from mice. NOS III could be detected in cochlea and vestibular ganglion cells, in nerve fibres, in outer hair cells of the cochlear and in the sensory epithelium of the maculae. Also, the spiral ligament and the limbus epithelium was positive to NOS III. NOS I was found in the sensory epithelium of the maculae and cristae ampullares, outer and inner hair cells of the cochlea, in nerve fibres and in ganglion cells. In contrast to that NOS II could not be detected at all. Furthermore, a strong NOS I immunoreaction was displayed on the endosteum of the bone, while the periosteum was lacking of NOS. NOS detection was accompanied by immunoreactivity to sGC. The findings imply that NOS I and III-generated NO is involved in neurotransmission and other regulative processes in the vestibulocochlear system.

The effects of L-NAME on vestibular compensation and NOS activity in the vestibular nucleus, cerebellum and cortex of the guinea pig.

Nitric oxide (NO) has been implicated in the processes by which animals recover from peripheral vestibular damage ('vestibular compensation'). However, few data exist on the dose-response effects of systemic administration of the nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), on the vestibular compensation process. The aim of this study was to investigate the effects on compensation of 5, 10, 50 or 100 mM L-NAME administered by s.c osmotic minipump for 50 h following unilateral vestibular deafferentation (UVD) in guinea pig, either commencing the drug treatment at 4 h pre-UVD or at the time of the UVD (i.e., post-UVD). Post-UVD treatment with L-NAME, at any of the four concentrations used, had no effect on the compensation of spontaneous nystagmus (SN), yaw head tilt (YHT) or roll head tilt (RHT). By contrast, pre-UVD treatment with 100 mM L-NAME resulted in a significant decrease in SN frequency (P<0.05) and a change in the rate of its compensation (P<0.0005). Pre-UVD L-NAME resulted in a significant increase in the overall magnitude of YHT (P<0.005); however, post-hoc comparisons revealed no significant differences between any specific L-NAME and vehicle groups. Pre-UVD L-NAME had no effect on RHT at any concentration. Analysis of NOS activity in the pre-UVD L-NAME treatment groups at 50 h post-UVD showed that only 100 mM L-NAME resulted in a significant decrease in NOS activity in the contralateral medial vestibular nucleus (MVN)/prepositus hypoglossi (PH) (P<0.05) and that NOS activity in the ipsilateral MVN/PH was not significantly affected. However, NOS activity was significantly inhibited in the bilateral cerebellum and cortices for several concentrations of L-NAME. These results suggest that pre-UVD systemic administration of L-NAME can significantly increase the rate of SN compensation in guinea pig and that this effect is correlated with inhibition of NOS activity in several regions of the CNS.

Development of vestibular function: biochemical, morphological and electronystagmographical assessment in the rat.

Glutamate decarboxylase and choline acetyltransferase were measured in homogenated ampullar cristae of rats during development from postnatal day 13 to 60 to determine changes in levels of these enzymes during early postnatal development. Afferent and efferent innervation of the hair cells of the developing cristae were studied using electron microscopy. In parallel, groups of rats, postrotatory nystagmus were used to assess the development of semicircular canal function during the same time interval. The level of glutamate decarboxylase was high on postnatal day 15 and did not change notably over the remaining days to day 60. Activity of choline acetyltransferase was nearly absent at day 15, but reached levels seen in mature animals by day 17, and remained almost unchanged thereafter. In contrast, as revealed by electronmicroscopy, afferent and efferent innervation appeared to be mature by day 8. Postrotatory nystagmus presented the adult-like features from day 19 onward. According to these results, a role for glutamate decarboxylase in afferent transmission is suggested by the parallel development of levels of glutamate decarboxylase and afferent innervation of the ampullary cristae. The finding of a similar time course of development of choline acetyltransferase levels and postrotatory nystagmus suggests that a cholinergic efferent innervation is involved in the onset of vestibular-ocular function.

Recovery of vestibular function in young guinea pigs after streptomycin treatment. Glutamate decarboxylase activity and nystagmus response assessment.

Fifty-day streptomycin (STP) treatment in guinea pigs causes specific vestibular hair cell (VHC) types I and II (HCI; HCII) degeneration, depletion of glutamate decarboxylase (GAD) and a gradual disappearance of postrotatory nystagmus response (PRNR), which is a sign of vestibular function alteration. In order to look for a possible spontaneous reversibility and its time course guinea pigs receiving 300 mg/kg STP daily were monitored for PRNR and vestibular GAD loss. Once PRNR was lost, STP was interrupted and the animal was allowed to recover; at the time that PRNR was completely reestablished, vestibular GAD was measured. PRNR was lost within 22-25 days of STP treatment. Vestibular GAD showed a loss that, with time of treatment, gave two slopes: a fast decrement (45% in 20 days) and a slow one (40% in the remaining 30). Stopping of the STP treatment after 22-25 days and animal recovery resulted in the return of both PRNR and GAD activity 22 days after STP stoppage. These results suggest two STP-susceptible GAD-containing VHC populations, one more sensitive than the other, possibly HCI followed by hair cell II (HCII). As hypothetic HCI loss and PRNR disappearance is simultaneous, the important role of the former for vestibular function could be inferred. Interruption of STP treatment after PRNR loss results in a long range restoration of both GAD activity and vestibular function, and thus recovery of HCI, the first evidence of its occurrence in a mammalian vestibule, could be suggested. The intimate mechanism of this recovery remains to be seen.

Immunohistochemical detection of vascular endothelial growth factor (VEGF) and VEGF-receptors Flt-1 and KDR/Flk-1 in the vestibule of guinea pigs.

Vascular endothelial growth factor (VEGF), known as an endothelial cell-specific mitogen, has been reported to be linked also to the NO/cGMP-pathway, which has been notified in the inner ear. Up to now, VEGF has not yet been described in the inner ear. We performed immunohistochemical analysis using specific antibodies to VEGF and to both known VEGF-receptors Flt-1 and KDR/Flk-1 on paraffin-sections of temporal bones from guinea pigs (n=5). Immunoreactivity of VEGF, Flt-1 and KDR/Flk-1 was detectable in a subpopulation of vestibular ganglion cells. VEGF could be found also in the endothelium of blood vessels, in fibrocytes of the lamina propria and in the neuroepithelium. Strong immuno-labelling to Flt-1 was evident in nerve fibres, vascular endothelium and in the neuroepithelium. Fibrocytes, endothelium of blood vessels, supporting cells and calyces in the sensory epithelium revealed immunoreactivity to KDR/Flk-1. These findings give evidence that VEGF, Flt-1 and KDR/Flk-1 are constitutively expressed in the vestibule.

Onset and development of neuron-specific enolase immunoreactivity in the peripheral vestibular system of the mouse.

The development of neuron-specific enolase (NSE) immunoreactivity in mouse sensory and ganglion vestibular cells was studied from gestation day 14 to adulthood. NSE staining appeared sequentially in these structures with a pattern that closely paralled their maturation sequences. The onset of NSE reactivity, at gestation day 15, in ganglion cells was concomitant with the formation of contacts between the afferent fibers and sensory cells, and was observed in hair cells, at gestation day 17, during the period in which the first synaptic structures form. The development of NSE staining in the cristae revealed an apex-base and axial gradients of maturation.

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.

Expression of inducible nitric oxide-synthase in the vestibular system of hydropic guinea pigs.

Immunohistochemical investigations of the guinea pig vestibular system, using a specific antibody to the inducible isoform of NO-synthase (iNOS/NOS II), have been performed 3 weeks after surgical closure of the right endolymphatic duct (n = 7). Endolymphatic hydrops (ELH) of the right temporal bone became evident by excavation of the Reissner's membrane in all seven animals. Those animals revealed iNOS-expression in ganglion cells, in the wall of blood vessels and in nerve fibers of the right vestibular system, while the corresponding left temporal bones and temporal bones of non-operated controls (n = 6) as well as of sham-operated animals (n = 3) did not show any iNOS-positive structures. iNOS-generated NO could be involved in the pathophysiology of vestibular dysfunction in Meniere's disease.

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.

Immunolocalization of Na+,K(+)-ATPase and carbonic anhydrase in the gerbil's vestibular system.

The distribution of two ion transport enzymes in the vestibular system was investigated immunocytochemically. Immunostaining demonstrated abundant Na+,K(+)-ATPase in the basolateral plasmalemma of all dark cells and of cuboidal (transitional) cells bordering maculae and planum semilunatum cells bordering cristae. Na+,K(+)-ATPase was also present in nerve terminals impinging on vestibular hair cells and around nerve fibers and ganglion cells. Na+,K(+)-ATPase containing cells with fine intertwining processes were found within the perilymphatic stroma beneath maculae and cristae. These cells and interspersed nerves form a distinct, highly cellular plate that lies under neurosensory epithelium selectively. The catalytic alpha subunit of Na+,K(+)-ATPase in vestibular epithelia differs antigenically from the alpha subunit in nerves and from the alpha subunit in salivary gland and renal epithelium. Carbonic anhydrase (CA) isozyme II was localized in the apex of all supporting cells in neurosensory epithelia. In contrast, CA II immunostaining varied in vestibular dark cells showing heterogeneity in ion transport activity among these cells. Immunostaining evidenced CA II also in perilymphatic stromal cells which were presumably fibroblastic in nature and which correspond in location with the Na+,K(+)-ATPase positive cells under the vestibular neurosensory epithelium.

Cholinesterase activity in vestibular organs of young and old mice.

The activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were studied by histochemical methods in the semicircular canal end organs, the utricle and the saccule of young and old mice. AChE was located on the plasma membrane of efferent nerve terminals beneath vestibular hair cells, and along the basement membrane. In the ampulla, stained efferent terminals were more prevalent on the slopes of the crista than in the central region. In all organs examined, there were no discernible differences in AChE activity between young and old mice. BChE activity was observed in the epithelial light cells and supporting cells of the saccule, utricle, and ampulla. Its distribution was similar in both young and old mice in the ampulla, but decreased significantly with age in the utricle. Preliminary data suggest that BChE activity is also weak in old saccular supporting cells. Unlike the utricle, old saccular light cells retained intense BChE activity.