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.

Characteristics of laser stimulation by near infrared pulses of retinal and vestibular primary neurons.

BACKGROUND AND OBJECTIVE: The optical stimulation of neurons from pulsed infrared lasers has appeared over the last years as an alternative to classical electric stimulations based on conventional electrodes. Laser stimulation could provide a better spatial selectivity allowing single-cell stimulation without prerequisite contact. In this work we present relevant physical characteristics of a non-lethal stimulation of cultured mouse vestibular and retinal ganglion neurons by single infrared laser pulses. STUDY DESIGN/MATERIALS AND METHODS: Vestibular and retinal ganglion neurons were stimulated by a 100-400 mW pulsed laser diode beam (wavelengths at 1,470, 1,535, 1,875 nm) launched into a multimode optical fiber positioned at a few hundred micrometers away from the neurons. Ionic exchange measurements at the neuron membrane were achieved by whole-cell patch-clamp recordings. Stimulation and damage thresholds, duration and repetition rate of stimulation and temperature were investigated. RESULTS: All three lasers induced safe and reproducible action potentials (APs) on both types of neurons. The radiant exposure thresholds required to elicit APs range from 15 ± 5 to 100 ± 5 J cm(-2) depending on the laser power and on the pulse duration. The damage thresholds, observed by a vital dye, were significantly greater than the stimulation thresholds. In the pulse duration range of our study (2-30 milliseconds), similar effects were observed for the three lasers. Measurements of the local temperature of the neuron area show that radiant exposures required for reliable stimulations at various pulse durations or laser powers correspond to a temperature increase from 22 °C (room temperature) to 55-60 °C. Stimulations by laser pulses at repetition rate of 1, 2, and 10 Hz during 10 minutes confirmed that the neurons were not damaged and were able to survive such temperatures. CONCLUSION: These results show that infrared laser radiations provide a possible way to safely stimulate retinal and vestibular ganglion neurons. A similar temperature threshold is required to trigger neurons independently of variable energy thresholds, suggesting that an absolute temperature is required.

Control of hair cell excitability by vestibular primary sensory neurons.

In the rat utricle, synaptic contacts between hair cells and the nerve fibers arising from the vestibular primary neurons form during the first week after birth. During that period, the sodium-based excitability that characterizes neonate utricle sensory cells is switched off. To investigate whether the establishment of synaptic contacts was responsible for the modulation of the hair cell excitability, we used an organotypic culture of rat utricle in which the setting of synapses was prevented. Under this condition, the voltage-gated sodium current and the underlying action potentials persisted in a large proportion of nonafferented hair cells. We then studied whether impairment of nerve terminals in the utricle of adult rats may also affect hair cell excitability. We induced selective and transient damages of afferent terminals using glutamate excitotoxicity in vivo. The efficiency of the excitotoxic injury was attested by selective swellings of the terminals and underlying altered vestibular behavior. Under this condition, the sodium-based excitability transiently recovered in hair cells. These results indicate that the modulation of hair cell excitability depends on the state of the afferent terminals. In adult utricle hair cells, this property may be essential to set the conditions required for restoration of the sensory network after damage. This is achieved via re-expression of a biological process that occurs during synaptogenesis.

Ultrastructural changes in otoconia of osteoporotic rats.

The etiology of benign paroxysmal positional vertigo (BPPV) remains obscure in many cases and women are affected more often than men. A recent prospective study, performed in women >50 years of age suffering from recurrent BPPV, showed associated osteopenia or osteoporosis in a large percentage of these patients. These results suggested the possible relationship between recurrent BPPV and a decreased fixation of calcium in bone in women >50 years. To test this hypothesis, an experimental study was performed in adult female rats. Utricular otoconia of female rats in which osteopenia/osteoporosis was induced by bilateral ovariectomy (OVX) were compared to those of sham-operated adult females rats (SHAM), as control group. FIRST STUDY: The morphology of theutricles of OVX and SHAM rats was analyzed with scanning electron microscopy. In osteopenic/osteoporotic rats, the density of otoconia (i.e. the number of otoconia per unit area) was decreased (p = 0.036)and their size was increased (p = 0.036) compared to the control group. SECOND STUDY: To test the role of calcium turnover in such morphological changes, utricular otoconia of 2 other groups of OVX and SHAM rats, previously injected with calcein subcutaneously, were examined by conventional and epifluorescence microscopy. In epifluorescence microscopy, labeling with calcein showed no significant fluorescence in either group. This finding was interpreted as a lack of external calcium turnover into otoconia of adult female rats. The ultrastructural modifications of otoconia in osteopenic/osteoporotic female adult rats as well as the role of estrogenic receptors in the inner ear are discussed. The possible pathophysiological mechanisms which support the relationship between recurrent BPPV in women and the disturbance of the calcium metabolism of osteopenia/osteoporosis are debated.

Trimetazidine modulates AMPA/kainate receptors in rat vestibular ganglion neurons.

Trimetazidine (1[2,3,4-trimethoxy-benzyl] piperazine, 2 HCl) is an anti-ischemic agent frequently administered as a prophylactic treatment for episodes of angina pectoris and chorioretinal disturbances. It is also employed as a symptomatic treatment of vertigo but its mechanism of action is yet to be defined. Using Fura-2 fluorescence photometry and whole-cell patch-clamp recordings we investigated the effect of trimetazidine on the [Ca(2+)](i) and current responses induced by the application of non-N-methyl-D-aspartate (NMDA) receptor agonists on low density vestibular ganglion neuronal cultures explanted from 3 day s postnatal rats. Trimetazidine blocked the [Ca(2+)](i) and current responses induced by 100 microM applications of both kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). These responses were dependent on external Ca(2+) and were blocked by the voltage-dependent Ca(2+) channel blockers Ni(2+) and Cd(2+) . Trimetazidine only acts on the AMPA/kainate receptors and had no effect on K(+)-induced depolarizations. Dose-dependent curves were obtained for the inhibition by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and trimetazidine (IC(50) 7 microM and 0.7 microM) of kainate stimulations. After AMPA stimulation, dose-response inhibition curves showed an IC(50) of 3 microM for CNQX and 25 microM for trimetazidine. These results indicate that trimetazidine could be a potent antagonist of AMPA/kainate receptors in vestibular ganglion neurons. This may explain the protective role of trimetazidine in the inner ear suggesting an anti-excitotoxic activity.

Ocsyn and mitochondrial-canalicular complexes in vestibular hair cells.

Ocsyn, a syntaxin-interacting protein characterized by Safieddine et al. [Safieddine, S., Ly, C.D., Wang, Y.-X., Kachar, B., Petralia, R.S., Wenthold, R.J., 2002. Ocsyn, a novel syntaxin-interacting protein enriched in the subapical region of inner hair cells. Mol. Cell. Neurosci., 20, 343-353] in the guinea pig organ of Corti was primarily identified in organelles located at the subapical region of inner hair cells. They proposed that in cochlear inner hair cells, ocsyn was involved in protein trafficking associated to recycling endosomes. Ocsyn happens to be highly homologous to syntabulin with an almost identical syntaxin-binding domain. Syntabulin is believed to attach syntaxin-containing vesicles to kinesin for their axonal transport along microtubules. The present study shows the distribution of ocsyn in guinea pig and rat vestibular hair cells using immunocytochemistry and confocal microscopy. Ocsyn was characterized by intense immunolabeled spots distributed exclusively in type I and II vestibular hair cells. The subcuticular region under the cuticular plate exhibited particularly densely packed spots. In the neck region of the sensory cells, where microtubules are abundant, there was no colocalization of ocsyn and alpha-tubulin. Ocsyn labeled spots were also present in the medial and basal hair cell regions, particularly in the supranuclear and infranuclear regions. Mitochondria are particularly numerous in these three regions (subcuticular, supranuclear and infranuclear). Double labeling of ocsyn and cytochrome c showed that ocsyn was present in the same zones that mitochondria. This, together with the great similarity of ocsyn and syntabulin, suggest that, akin to syntabulin, ocsyn is involved in addressing organelles. We propose that ocsyn is involved in the formation of the canalicular-mitochondrial complexes depicted by Spicer et al. [Spicer, S.S., Thomopoulos, G.N., Schulte, B.A., 1999. Novel membranous structures in apical and basal compartments of inner hear cells. J. Comp. Neurol., 409, 424-437].

Correlation between afferent rearrangements and behavioral deficits after local excitotoxic insult in the mammalian vestibule: a rat model of vertigo symptoms.

Damage to inner ear afferent terminals is believed to result in many auditory and vestibular dysfunctions. The sequence of afferent injuries and repair, as well as their correlation with vertigo symptoms, remains poorly documented. In particular, information on the changes that take place at the primary vestibular endings during the first hours following a selective insult is lacking. In the present study, we combined histological analysis with behavioral assessments of vestibular function in a rat model of unilateral vestibular excitotoxic insult. Excitotoxicity resulted in an immediate but transient alteration of the balance function that was resolved within a week. Concomitantly, vestibular primary afferents underwent a sequence of structural changes followed by spontaneous repair. Within the first two hours after the insult, a first phase of pronounced vestibular dysfunction coincided with extensive swelling of afferent terminals. In the next 24 h, a second phase of significant but incomplete reduction of the vestibular dysfunction was accompanied by a resorption of swollen terminals and fiber retraction. Eventually, within 1 week, a third phase of complete balance restoration occurred. The slow and progressive withdrawal of the balance dysfunction correlated with full reconstitution of nerve terminals. Competitive re-innervation by afferent and efferent terminals that mimicked developmental synaptogenesis resulted in full re-afferentation of the sensory epithelia. By deciphering the sequence of structural alterations that occur in the vestibule during selective excitotoxic impairment, this study offers new understanding of how a vestibular insult develops in the vestibule and how it governs the heterogeneity of vertigo symptoms.

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.

Symptomatic treatment of vestibular deficits: therapeutic potential of histamine H4 receptors.

Vestibular disorders display high prevalence and can severely impact the daily life. However, pharmacological options that would efficiently relieve the vertigo symptoms without side effects are still lacking. In the present review we briefly review the common history of histamine receptor modulation and the pharmacological therapy of vestibular disorders. We also discuss the recent demonstration of Histamine H4 Receptor mRNAs expression in Scarpa's ganglion of mammal and the potential use of specific H4R antagonists as vestibulomodulators. Additional original data confirm the expression of H4R proteins in the rat vestibular primary neurons, the neuromodulatory properties of specific H4R antagonists in vitro (inhibition of vestibular neuron excitability) as well as their efficacy to decrease vestibular deficits induced in different in animal models.

Plasticity of Scarpa's Ganglion Neurons as a Possible Basis for Functional Restoration within Vestibular Endorgans.

In a previous study, we observed spontaneous restoration of vestibular function in young adult rodents following excitotoxic injury of the neuronal connections within vestibular endorgans. The functional restoration was supported by a repair of synaptic contacts between hair cells and primary vestibular neurons. This process was observed in 2/3 of the animals studied and occurred within 5 days following the synaptic damage. To assess whether repair capacity is a fundamental trait of vestibular endorgans and to decipher the cellular mechanisms supporting such a repair process, we studied the neuronal regeneration and synaptogenesis in co-cultures of vestibular epithelia and Scarpa's ganglion from young and adult rodents. We demonstrate that, under specific culture conditions, primary vestibular neurons from young mice or rats exhibit robust ability to regenerate nervous processes. When co-cultured with vestibular epithelia, primary vestibular neurons were able to establish de novo contacts with hair cells. Under the present paradigm, these contacts displayed morphological features of immature synaptic contacts. Preliminary observations using co-cultures of adult rodents suggest that this reparative capacity remained in older mice although to a lesser extent. Identifying the basic mechanisms underlying the repair process may provide a basis for novel therapeutic strategies to restore mature and functional vestibular synaptic contacts following damage or loss.

Distinct population of hair cell progenitors can be isolated from the postnatal mouse cochlea using side population analysis.

In mammals, the permanence of hearing loss is due mostly to the incapacity of the cochlea to replace lost mechano-receptor cells (i.e., hair cells [HCs]). The generation of new HCs from a renewable source of progenitors is a principal requirement for developing a cell therapy within this sensory organ. A subset of stem cells, termed side population (SP), has been identified in several tissues of mammals. The ATP-binding cassette transporter Abcg2/Bcrp1 contributes to the specification of the SP phenotype and is proposed as a universal marker for stem/progenitor cells. A defining character of these SP cells is a high efflux capacity for Hoechst dye. Here, we demonstrate that Abcg2 transporter is expressed with two other stem/progenitor cell markers (i.e., Nestin and Musashi1) in distinct and overlapping domains of the supporting cells within the postnatal cochlea. We have developed and describe a fluorescence-activated cell sorting (FACS) technique that enables the purification of a discrete subpopulation of SP-supporting cells from the early postnatal mouse cochlea based on their ability to exclude Hoechst dye. These FACS-isolated cells can divide and express markers of stem/progenitor cells such as Abcg2, a determinant of the SP phenotype, and Musashi1, a neural stem/progenitor cell marker. These markers can differentiate cells expressing markers of HCs and supporting cells in vitro. Our observation that these SP cells are capable of differentiating into HC-like cells implies a possible use for such cells (i.e., the replacement of lost auditory HCs within damaged cochlea).

Vestibular Schwann cells are a distinct subpopulation of peripheral glia with specific sensitivity to growth factors and extracellular matrix components.

Vestibular nerve Schwann cells are predisposed to develop schwannoma. While knowledge concerning this condition has greatly improved, little is known about properties of normal vestibular Schwann cells. In an attempt to understand this predisposition, we evaluated cell density regulation and proliferative features of these cells taken from 6-day-old rats. Data were compared to those obtained with sciatic Schwann cells. In both vestibular and sciatic 7-day-old cultures, Schwann cells appear as bipolar or flattened cells. However, sciatic and vestibular cells greatly differ in other aspects: on poly-L-lysine coating, sciatic cells specifically synthesize myelin basic protein, while expression of P0 mRNAs is restricted to some vestibular cells. Laminin increases sciatic cell density but not that of vestibular cells. Fibronectin selectively enhances the proliferation of vestibular Schwann cells and lacks an effect on sciatic ones. Comparison of cell density changes between sciatic and vestibular cells shows that they are sensitive to two different sets of growth factors. Progesterone and FGF-2 combined with forskolin selectively enhance the cell density of sciatic glia, while IGF-1 and GDNF specifically increase vestibular cell density. Furthermore, BrdU incorporation assays indicate that GDNF is also a mitogen for vestibular cells. Altogether, vestibular Schwann cells display phenotypic features and responsiveness to exogenous signals that are significantly different from sciatic Schwann cells, suggesting that vestibular glia form a subpopulation of Schwann cells.