Critical roles of transitional cells and Na/K-ATPase in the formation of vestibular endolymph.

The mechanotransduction of vestibular sensory cells depends on the high endolymphatic potassium concentration ([K+]) maintained by a fine balance between K+ secretion and absorption by epithelial cells. Despite the crucial role of endolymph as an electrochemical motor for mechanotransduction, little is known about the processes that govern endolymph formation. To address these, we took advantage of an organotypic rodent model, which regenerates a genuine neonatal vestibular endolymphatic compartment, facilitating the determination of endolymphatic [K+] and transepithelial potential (Vt) during endolymph formation. While mature Vt levels are almost immediately achieved, K+ accumulates to reach a steady [K+] by day 5 in culture. Inhibition of sensory cell K+ efflux enhances [K+] regardless of the blocker used (FM1.43, amikacin, gentamicin, or gadolinium). Targeting K+ secretion with bumetanide partially and transiently reduces [K+], while ouabain application and Kcne1 deletion almost abolishes it. Immunofluorescence studies demonstrate that dark cells do not express Na-K-2Cl cotransporter 1 (the target of bumetanide) in cultured and young mouse utricles, while Na/K-ATPase (the target of ouabain) is found in dark cells and transitional cells. This global analysis of the involvement of endolymphatic homeostasis actors in the immature organ (1) confirms that KCNE1 channels are necessary for K+ secretion, (2) highlights Na/K-ATPase as the key endolymphatic K+ provider and shows that Na-K-2Cl cotransporter 1 has a limited impact on K+ influx, and (3) demonstrates that transitional cells are involved in K+ secretion in the early endolymphatic compartment.

Tmprss3, a transmembrane serine protease deficient in human DFNB8/10 deafness, is critical for cochlear hair cell survival at the onset of hearing.

Mutations in the type II transmembrane serine protease 3 (TMPRSS3) gene cause non-syndromic autosomal recessive deafness (DFNB8/10), characterized by congenital or childhood onset bilateral profound hearing loss. In order to explore the physiopathology of TMPRSS3 related deafness, we have generated an ethyl-nitrosourea-induced mutant mouse carrying a protein-truncating nonsense mutation in Tmprss3 (Y260X) and characterized the functional and histological consequences of Tmprss3 deficiency. Auditory brainstem response revealed that wild type and heterozygous mice have normal hearing thresholds up to 5 months of age, whereas Tmprss3(Y260X) homozygous mutant mice exhibit severe deafness. Histological examination showed degeneration of the organ of Corti in adult mutant mice. Cochlear hair cell degeneration starts at the onset of hearing, postnatal day 12, in the basal turn and progresses very rapidly toward the apex, reaching completion within 2 days. Given that auditory and vestibular deficits often co-exist, we evaluated the balancing abilities of Tmprss3(Y260X) mice by using rotating rod and vestibular behavioral tests. Tmprss3(Y260X) mice effectively displayed mild vestibular syndrome that correlated histologically with a slow degeneration of saccular hair cells. In situ hybridization in the developing inner ear showed that Tmprss3 mRNA is localized in sensory hair cells in the cochlea and the vestibule. Our results show that Tmprss3 acts as a permissive factor for cochlear hair cells survival and activation at the onset of hearing and is required for saccular hair cell survival. This mouse model will certainly help to decipher the molecular mechanisms underlying DFNB8/10 deafness and cochlear function.

M-like K+ currents in type I hair cells and calyx afferent endings of the developing rat utricle.

Type I vestibular hair cells have large K+ currents that, like neuronal M currents, activate negative to resting potential and are modulatable. In rodents, these currents are acquired postnatally. In perforated-patch recordings from rat utricular hair cells, immature hair cells [younger than postnatal day 7 (P7)] had a steady-state K+ conductance (g(-30)) with a half-activation voltage (V1/2) of -30 mV. The size and activation range did not change in maturing type II cells, but, by P16, type I cells had added a K conductance that was on average fourfold larger and activated much more negatively. This conductance may comprise two components: g(-60) (V1/2 of -60 mV) and g(-80) (V1/2 of -80 mV). g(-80) washed out during ruptured patch recordings and was blocked by a protein kinase inhibitor. M currents can include contributions from KCNQ and ether-a-go-go-related (erg) channels. KCNQ and erg channel blockers both affected the K+ currents of type I cells, with KCNQ blockers being more potent at younger than P7 and erg blockers more potent at older than P16. Single-cell reverse transcription-PCR and immunocytochemistry showed expression of KCNQ and erg subunits. We propose that KCNQ channels contribute to g(-30) and g(-60) and erg subunits contribute to g(-80). Type I hair cells are contacted by calyceal afferent endings. Recordings from dissociated calyces and afferent endings revealed large K+ conductances, including a KCNQ conductance. Calyx endings were strongly labeled by KCNQ4 and erg1 antisera. Thus, both hair cells and calyx endings have large M-like K+ conductances with the potential to control the gain of transmission.

Weightlessness affects cytoskeleton of rat utricular hair cells during maturation in vitro.

The aim of this study was to investigate whether an altered gravitational environment affected the phenotype of vestibular hair cells during maturation. We developed, using an automated incubator, a 3D culture of utricles from newborn rats. These cultures were subjected to weightlessness for 1 or 3 days, and then compared with control cultures developed in natural and induced 1G gravity. Immunocytochemistry for alpha-tubulin and calretinin revealed disorganisation of the microtubules and a loss of hair cell shape in cells subjected to weightlessness during maturation. We conclude that the lack of gravitational strain affected cytoskeletal dynamics, resulting in loss of the specific morphological phenotype of the cells.

Differential impact of hypergravity on maturating innervation in vestibular epithelia during rat development.

Over the past decades, the new opportunity of space flights has revealed the importance of gravity as a mechanical constraint for terrestrial organisms as well as its influence on the somatosensory system. The lack of gravitational reference in orbital flight induces changes in equilibrium, with major modifications involving neuromorphological and physiological adaptations. However, few data have illustrated the putative effect of gravity on sensory vestibular epithelial development. We asked if gravity, the primary stimulus of utricles could act as an epigenetic factor. As sensorial deprivation linked to weightlessness is technically difficult, we used a ground-based centrifuge to increase the gravitational vector, in order to hyperstimulate the vestibule. In this study, 3 days after mating, pregnant females were submitted to hypergravity, 2 g (HG). Their embryos were raised, born and postnatally developed under HG. The establishment of connections between primary vestibular afferent neurons and hair cells in the utricle of these young rats was followed from birth to postnatal day 6 (PN6) and compared to embryos developed in normogravity (NG): Immunocytochemistry for neurofilaments and microvesicles revealed the differential effects of gravity on the late neuritogenic and synaptogenic processes in utricles. Taking type I hair cell innervation as a criterion of maturation, we found that primary afferent fibres reached the vestibular epithelium and enveloped hair cells in the same way, both under NG and HG. Thus, this phenomenon of leading growth cones to their epithelial target appears to be dependent on intrinsic genetic properties and not on an external stimulus. In contrast, the maturation of connection processes between type 1 hair cells and the afferent calyx, concerning specifically the microvesicles at their apex, was delayed under HG. Therefore, gravity appears to be an epigenetic factor influencing the late maturation of utricles. These differential effects of altered gravity on the development of the vestibular epithelium are discussed.

Developmental changes in two voltage-dependent sodium currents in utricular hair cells.

Two kinds of sodium current (I(Na)) have been separately reported in hair cells of the immature rodent utricle, a vestibular organ. We show that rat utricular hair cells express one or the other current depending on age (between postnatal days 0 and 22, P0-P22), hair cell type (I, II, or immature), and epithelial zone (striola vs. extrastriola). The properties of these two currents, or a mix, can account for descriptions of I(Na) in hair cells from other reports. The patterns of Na channel expression during development suggest a role in establishing the distinct synapses of vestibular hair cells of different type and epithelial zone. All type I hair cells expressed I(Na,1), a TTX-insensitive current with a very negative voltage range of inactivation (midpoint: -94 mV). I(Na,2) was TTX sensitive and had less negative voltage ranges of activation and inactivation (inactivation midpoint: -72 mV). I(Na,1) dominated in the striola at all ages, but current density fell by two-thirds after the first postnatal week. I(Na,2) was expressed by 60% of hair cells in the extrastriola in the first week, then disappeared. In the third week, all type I cells and about half of type II cells had I(Na,1); the remaining cells lacked sodium current. I(Na,1) is probably carried by Na(V)1.5 subunits based on biophysical and pharmacological properties, mRNA expression, and immunoreactivity. Na(V)1.5 was also localized to calyx endings on type I hair cells. Several TTX-sensitive subunits are candidates for I(Na,2).