Three-dimensional culture of newborn rat utricle using an extracellular matrix promotes formation of a cyst.

The vestibule is the end organ devoted to sensing of head movements in space. To function properly, its mechano-receptors require the presence of a unique apical extracellular medium, the endolymph. Numerous studies have elucidated the mechanisms involved in the production and homeostasis of this unique medium and the responses of sensory cells to stimulation. However, anatomical constraints have prevented direct and simultaneous studies of their relationships. The aim of this study was the development of an in vitro model that would allow concomitant investigations on maturation and physiological properties of both the hair cells and their endolymphatic compartment. A three-dimensional (3D) culture of newborn rat utricles using an extracellular matrix sustaining 3D cellular growth was developed during 3, 6, or 10 days in vitro (DIV). Using morphological and electrophysiological techniques, we describe the de novo formation of a cyst. It was composed of the sensory epithelium and non-sensory cells-canalar, dark and intermediate cells-that polarized so that their apical surface faced its lumen. During the time of culture, the utricular potential (UP) was steady (-1.1+/-5.0 mV) in oxygenated condition, while in anoxia, the UP significantly decreased to -8.4+/-1.0 mV at 8 DIV. Over the same period, the K+ concentration in the cyst increased up to 86.1+/-33.9 mM (versus 5.6+/-1.5 mM in the bath). These observations indicated that the mechanisms generating the UP and the K-secretory activity were functional at this stage. Concomitantly, the hair cells acquired mature and functional properties: the type 1 and type 2 phenotypes, a mean resting membrane potential of -68.1+/-4.6 mV and typical electrophysiological responses. This preparation provides a powerful means to simultaneous access the hair cells and their endolymphatic compartment, with the possibility to use multi-technical approaches to investigate their interdependent relationships.

Contribution of the plasmalemma to Ca2+ homeostasis in hair cells.

Calcium influx through transduction channels and efflux via plasmalemmal Ca(2+)-ATPases (PMCAs) are known to contribute to calcium homeostasis and modulate sensory transduction in vertebrate hair cells. To examine the relative contributions of apical and basolateral pathways, we analyzed the calcium dynamics in solitary ciliated and deciliated guinea pig type I and type II vestibular hair cells. Whole-cell patch-clamp recordings demonstrated that these cells had resting potentials near -70 mV and could be depolarized by 10-20 mV by superfusion with high potassium. Fura-2 measurements indicated that ciliated type II cells and deciliated cells of either type had low basal [Ca(2+)](i), near approximately 90 nm, and superfusion with high potassium led to transient calcium increases that were diminished in the presence of Ca(2+) channel blockers. In contrast, measurements of type I ciliated cells, hair cells with large calyceal afferents, were associated with a higher basal [Ca(2+)](i) of approximately 170 nm. High-potassium superfusion of these cells induced a paradoxical decrease in [Ca(2+)](i) that was augmented in the presence of Ca(2+) channel blockers. Optical localization of dihydropyridine binding to the kinocilium suggests that they contain L-type calcium channels, and as a result apical calcium influx includes a contribution from voltage-dependent ion channels in addition to entry via transduction channels localized to the stereocilia. Eosin block of PMCA significantly altered both [Ca(2+)](i) baseline and transient responses only in ciliated cells suggesting that, in agreement with immunohistochemical studies, PMCA is primarily localized to the bundles.

Ionic currents and current-clamp depolarisations of type I and type II hair cells from the developing rat utricle.

Ionic currents and the voltage response to injected currents were studied in an acutely dissected preparation of the rat utricle between birth and postnatal day 12 (PN12). Based upon morphological criteria, the sensory cells examined were divided into two classes, "type I" and "type 2 category," the latter of which may include some immature type I cells. The former group comprises a clearly defined electrophysiological population, with one large outwardly rectifying potassium conductance that is sensitive to 4-aminopyridine (4-AP), insensitive to tetraethylammonium (TEA) and displays voltage-dependent activation kinetics. In the absence of enzymatic dissociation procedures, and with the epithelium left largely intact, the mean half activation of this conductance was -30.3 mV at PN3, and -37.5 mV at PN12. At both stages it was almost entirely turned off at -74 mV. Omission of ATP from the intracellular solution appeared to prevent rundown of this conductance. Type II category hair cells formed a more heterogeneous population, exhibiting a distinct TEA-sensitive delayed rectifier potassium conductance; the rapidly activating and inactivating IA; an inward rectifier; and inward sodium currents at around PN3. Both cell types depolarised strongly in response to injected currents, with time courses reflecting the activation kinetics of their major outward conductances.

K+-dependence of Na+-Ca2+ exchange in type I vestibular sensory cells of guinea-pig.

The properties of the vestibular Na+-Ca2+ exchanger in mammalian type I vestibular sensory cells were studied using fura-2 fluorescence and immunocytochemical techniques. In the absence of external Na+, the activation of Na+-Ca2+ exchange in reverse mode required the presence of external K+ (K+o) and depended on K+o concentration. Alkali cations Rb+ and NH4+ but not Li+ or Cs+ substituted for K+o to activate the exchange. For pressure applications of 10 mm K+, the contribution of voltage-sensitive calcium channels to the increase in [Ca2+]i was < 15%. The dependence of the exchange on [K+]o was also recorded when the membrane potential was clamped using carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) and monensin ionophores. In these conditions, where there was no intracellular Na+, the increase in [Ca2+]i was completely blocked. These physiological results suggest that in reverse mode, Ca2+ entry is driven by both an outward transport of Na+ and an inward transport of K+. The dependence of the vestibular Na+-Ca2+ exchanger on K+ is more reminiscent of the properties of the retinal type Na+-Ca2+ exchanger than those of the more widely distributed cardiac type exchanger. Moreover, the immunocytochemical localization of both types of exchange proteins in the vestibular sensory epithelium confirmed the presence in the vestibular sensory cells of a Na+-Ca2+ exchanger which is recognized by an antibody raised against retinal type and not by an antibody raised against the cardiac type.

Potassium depolarization of mammalian vestibular sensory cells increases [Ca2+]i through voltage-sensitive calcium channels.

The existence of voltage-sensitive Ca2+ channels in type I vestibular hair cells of mammals has not been conclusively proven. Furthermore, Ca2+ channels present in type II vestibular hair cells of mammals have not been pharmacologically identified. Fura-2 fluorescence was used to estimate, in both cell types, intracellular Ca2+ concentration ([Ca2+]i) variations induced by K+ depolarization and modified by specific Ca2+ channel agonists and antagonists. At rest, [Ca2+]i was 90 +/- 20 nM in both cell types. Microperifusion of high-K+ solution (50 mM) for 1 s increased [Ca2+]i to 290 +/- 50 nM in type I (n = 20) and to 440 +/- 50 nM in type II cells (n = 10). In Ca2+-free medium, K+ did not alter [Ca2+]i. The specific L-type Ca2+ channel agonist, Bay K, and antagonist, nitrendipine, modified in a dose-dependent manner the K+-induced [Ca2+]i increase in both cell types with maximum effect at 2 microM and 400 nM, respectively. Ni2+, a T-type Ca2+ channel blocker, reduced K+-evoked Ca2+ responses in a dose-dependent manner. For elevated Ni2+ concentrations, the response was differently affected by Ni2+ alone, or combined to nitrendipine (500 nM). In optimal conditions, nitrendipine and Ni2+ strongly depressed by 95% the [Ca2+]i increases. By contrast, neither omega-agatoxin IVA (1 microM), a specific P- and Q-type blocker, nor omega-conotoxin GVIA (1 microM), a specific N-type blocker, affected K+-evoked Ca2+i responses. These results provide the first direct evidence that L- and probably T-type channels control the K+-induced Ca2+ influx in both types of sensory cells.

Novel monoacetoneglucose derivatives with calcium antagonist activity.

Ethers and thioethers of monosaccharides have been synthesised which show potent toxicity to mouse (LD50 > or = 4 g.kg-1 O.W. and 0.2 to 1.5 g.kg-1 I.P.W.). A study of calcium antagonist activity for the full series of compounds indicated that the activity was similar for both O- and S- ethers and maximum activities were observed for monoacetoneglucose ethers possessing carbon chain close to 8 carbons.

Calcium homeostasis in guinea pig type-I vestibular hair cell: possible involvement of an Na(+)-Ca2+ exchanger.

In type-I vestibular hair cells (VHCs), the mechanisms involved in intracellular calcium homeostasis have not yet been established. In order to investigate the involvement of an Na(+)-dependent ionic exchanger in the regulation of cytosolic free calcium concentration, we analyzed the effect of the removal of external sodium on the cytosolic concentration of calcium ions ([Ca2+]i), sodium ions ([Na+]i), and protons (pHi). These concentrations were measured in type-I VHCs isolated from guinea pig labyrinth, using Fura-2, sodium benzofuran isophtalate (SBFI), and 1,4 diacetoxy-2,3 dicyanobenzol (ADB) respectively. Complete replacement of Na+ in the superfusion solution with N-methyl-D-glucamine (NMDG+), reversibly increased [Ca2+]i by 276 +/- 89% (n = 46) and decreased [Na+]i by 23 +/- 6% (n = 14). Both responses were prevented by removing external Ca2+ or chelating internal Ca2+. This suggests the presence of coupled Ca2+ and Na+ transport. The [Ca2+]i increase evoked by Na(+)-free solution was reduced by about 55% with the application of amiloride derivatives and was totally abolished in the presence of high [Mg2+]o. No pHi variation was detected during [Na+]o reduction. In the absence of external K+, the Na(+)-free solution failed to induce [Ca2+]i increase; the readmission of external K+ restored the [Ca2+]i response. These results are consistent with a Na(+)-Ca2+ exchanger operating in reverse mode. An K+ dependence of this exchange is also suggested.

Transient increase in cytosolic free calcium evoked by repolarization in type I vestibular hair cells of rats.

Simultaneous whole-cell patch clamp and Fura-2 microfluorimetric recordings of membrane currents and intracellular free calcium concentration ([Ca2+]i) were made from type I vestibular hair cells isolated from cristae ampullares of adult rats. Cells held between -110 or -70 mV and depolarized up to -20 mV did not evoke any [Ca2+]i changes for any duration of the membrane depolarization (up to 3 s). Returning the membrane to repolarizing potential induced a transient [Ca2+]i increase. At the pulse break, an inward current was evoked. The [Ca2+]i increase and inward current amplitude were dependent on the duration and the amplitude of the previous depolarization. A liminar value of membrane depolarization of -55 +/- 3 mV (mean resting potential -62 +/- 7 mv) had to be applied to induce [Ca2+]i increase upon subsequent repolarization. [Ca2+]i response and inward current could not be evoked in calcium-free solution. Both responses were restored when calcium was added to the medium.

Cholinergic agonists increase intracellular calcium concentration in frog vestibular hair cells.

Acetylcholine (ACh) is usually considered to be the neurotransmitter of the efferent vestibular system. The nature and the localization of cholinergic receptors have been investigated on frog isolated vestibular hair cells (VHCs), by measuring variations of intracellular calcium concentration ([Ca2+]i), using calcium sensitive dye fura-2. Focal iontophoretic ACh (1 M, 300 nA.40 ms) application induced a rapid increase in [Ca2+]i, reaching a peak in 20 s and representing about 5-fold the resting level (from 61 +/- 6 to 320 +/- 26 nM). Applications of muscarinic agonists as methacholine and carbachol induced weaker calcium responses (from 78 +/- 25 to 238 +/- 53 nM) than the one obtained with ACh applications. These muscarinic agonists were efficient only in precise zones. Desensitization of muscarinic receptors to successive stimulations was significant. Perfusion of nicotine or 1,1-dimethyl-4-phenyl-piperazinium (DMPP), a nicotinic agonist, induced an increase in [Ca2+]i only in some cells (4/28 with DMPP). These results indicated the presence of cholinergic receptors on frog VHCs: muscarinic receptors were more responsive than nicotinic receptors. Presence of muscarinic and nicotinic receptors in the membrane of VHCs could indicate different modulations of VHCs activity mediated by [Ca2+]i and involving an efferent control which represents a central regulation of the vestibular afferent message.

Intracellular calcium variations evoked by mechanical stimulation of mammalian isolated vestibular type I hair cells.

The variations of intracellular free calcium concentration ([Ca2+]i) were recorded on-line from guinea-pig isolated vestibular sensory cells using a fura-2 fast fluorescent photometry system, during mechanical displacements of the hair bundle. Repetitive displacements of the hair bundle towards the kinocilium (positive stimulation 7 degrees, 300 ms, 2Hz for 10 s), revealed [Ca2+]i variations detectable only in the cuticular plate. [Ca2+]i increased from 105 to 145 nM. Single mechanical displacements of the hair bundle (7 degrees, 200 ms, 0.5 Hz) evoked increases of [Ca2+]i from 50 +/- 23 nM to 139 +/- 79 (n = 12). In the opposite direction, the mechanical stimulations (8 degrees, 400 ms, 0.5 Hz) evoked a decrease of [Ca2+]i from 68 +/- 17 nM to 37 +/- 12 nM (n = 8). The variations of [Ca2+]i detected in the cuticular plate during positive displacements of the hair bundle were reversibly abolished in the presence of 100 microM gentamicin and they could not be evoked in 0.1 mM calcium in the external medium. From these experiments, it has been concluded that the [Ca2+]i variations recorded in the cuticular plate were due to a limited entry of calcium ions through transduction channels localized in the hair bundle. The typical kinetics of variations of [Ca2+]i evoked during positive displacements of the hair bundle should account for the presence of strong calcium regulation systems in the hair bundle and cuticular plate.

The vestibular type I hair cells: a self-regulated system?

In this study the functional role of afferent nerve calyx surrounding the type I vestibular hair cells was investigated. Synaptic microvesicles were present at the apex of the calyx in the vestibular epithelium of human foetuses at 9 weeks from gestation. Whole cell clamped type I hair cells isolated from guinea pig epithelium presented active movements as shortening of the neck and tilting of the cuticular plate at the cessation of the depolarising step. These movements were calcium dependent. With the aim of establishing the kinetics of calcium influx during the cell depolarisation, intracellular free calcium rate variations were investigated by coupling cytofluorimetry technique with whole cell patch clamp. An increase of intracellular calcium was only observed at the repolarisation of type I hair cells. Thus, a regulatory short-loop is thought to exist to control adaptation phenomena at the upper part of the type I hair cell. It is suggested that this occurs through the release of a neurotransmitter from the apex of the afferent calyx.

Potassium currents in type II vestibular hair cells isolated from the guinea-pig's crista ampullaris.

Type II vestibular hair cells were isolated from cristae ampullares of guinea-pig and maintained in vitro for 2-3 h. Outward membrane currents were studied under whole-cell voltage-clamp conditions. Type II hair cells had resting potentials of about -45 mV. Depolarizing voltage steps from a holding potential of -80 or -90 mV induced time- and voltage-dependent outward currents which slowly decayed to a sustained level. Tail currents reversed at about -70 mV, indicating that the outward currents were mainly carried by potassium ions. The currents had an activation threshold around -50 mV. The transient component was completely removed by a depolarizing pre-pulse positive to -10 mV. While bath application of 4-aminopyridine (5 mM) reduced both components, extracellular tetraethylammonium (10 mM) or zero calcium preferentially diminished the sustained current. We conclude that at least two potassium conductances are present, a delayed rectifier with a relatively fast inactivation and a calcium-dependent potassium current. Depolarizing current injections induced an electrical resonance in the voltage responses, with a frequency of 25-100 Hz, larger currents causing higher frequencies.

Glutamate receptors on type I vestibular hair cells of guinea-pig.

Afferent nerve calyces which surround type I vestibular hair cells (VHCI) have recently been shown to contain synaptic-like vesicles and to be immunoreactive to glutamate antibodies. In order to understand the physiological significance of these observations, the presence of glutamate receptors on type I vestibular sensory cells has been investigated. The effect of excitatory amino acids applied by iontophoresis was examined by spectrofluorimetry using fura-2 sensitive dye. Glutamate application caused a rapid and transient increase in intracellular calcium concentration ([Ca2+]i), in a dose-dependent manner. The ionotropic glutamate receptors agonists N-methyl-D-aspartic acid (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and quisqualic acid (QA) induced an increase of [Ca2+]i. The NMDA receptor antagonist 2-amino-5-phosphonovaleric acid and the AMPA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione partially blocked the glutamate response, by 39 +/- 10 and 53 +/- 11% respectively. Metabotropic receptors were also revealed by the specific agonist trans-1-amino-cyclopentyl-1,3-dicarboxylate. The presence of different glutamate receptors on the VHCI membrane suggests two kinds of feedback. (i) At the base of the sensory cell, autoreceptors may locally control the synaptic transmission. (ii) At the apex, postsynaptic receptors may modulate sensory transduction from glutamate release at the upper part of the afferent nerve calyx. These feedbacks suggest presynaptic modulation of the vestibular hair cell response which could affect its sensitivity.

Voltage dependent reversible movements of the apex in isolated guinea pig vestibular hair cells.

Type I vestibular hair cells isolated from guinea pig were placed in the whole cell clamp configuration, and electrically stimulated by depolarizing voltage pulses. The voltage dependent reversible movements of the cell apex affected the length of the cell neck, the position of the cuticular plate, and the tilting and bending of the stereocilia. The cell neck shortened when the membrane was depolarized by 10 mV while cuticular plate and the stereocilia tilting did not begin until 20 mV. The shortening was 0.5 to 1 micron, and the cuticular plate tilting was up to 15 degrees for depolarization amplitudes of 20-40 mV. These movements were reversed within a few seconds. More complex, larger movements were induced by stronger depolarizations. The cuticular plate tilting and the hair bundle bending were always in the opposite direction to the kinocilium position. The small reversible movements of the mammalian type I vestibular hair cells are discussed in terms of mechanical adaptation processes and morphological features. It is suggested that such active movements of the vestibular hair cells occur in vivo.

Inward potassium rectifier current in type I vestibular hair cells isolated from guinea pig.

Large inward current activated by hyperpolarization was studied using whole cell patch clamp technique in type I vestibular hair cells of guinea pig. Near the resting membrane potential, at an holding potential of -60 mV (HP -60), this current increased with hyperpolarizing steps and showed time-dependent decay for steps below -80 mV. This current was progressively inactivated at more negative holding potential and was totally abolished at HP -90 mV. The underlying conductance was a K+ conductance as indicated by: (i) its dependence on the external potassium concentration; (ii) its tail currents, which reversed at about -90 mV in solutions with a normal gradient for K+ ions. Pharmacological studies revealed that external application of 4-aminopyridine (5 mM) reversibly blocked (95%) the total inward current, while external application of tetraethylammonium (10 mM) or cesium (2 mM) did not significantly affect the amplitude of this current. This potassium inward rectifier current could contribute to restoration of the resting membrane potential during negative stimulations.

Non-typical K(+)-current in cesium-loaded guinea pig type I vestibular hair cell.

Isolated guinea pig type I vestibular hair cells were voltage clamped at HP-110 mV in whole cell clamp configuration and depolarized up to +20 mV. Increasing depolarizations elicited large outward currents. These currents were replaced, in cesium-loaded cells, by inward/outward currents that reversed at membrane potentials between -55 and -30 mV. The reversal potential varied from cell to cell, and appeared to depend on the intracellular potassium cesium ratio. The current remaining in the presence of intracellular cesium was essentially due to a non-typical potassium conductance, which decreased in the presence of 4-AP and was blocked by 4-AP plus TEA. This current appeared as soon as the membrane was depolarized, showing the high potassium permeability of type I vestibular hair cells. A small part of this current was a strictly calcium inward current, sensitive to flunarizine, with a leakage component in the hyperpolarized state and a voltage component when the cell was depolarized.

Motile responses of isolated guinea pig vestibular hair cells.

Vestibular hair cells were isolated from the guinea pig vestibule by a micromechanical non-enzymatic procedure. Perfusion with 125 mM K+ solution induced irreversible slow shortening of the necks in 42.8% of the hair cells tested. Mechanical stimulation, creating a displacement of the hair bundle towards the kinocilium, induced either irreversible coiling or tilting of the neck of the cells, or reversible fast tilting of the cuticular plate (44.5% of tested cells). The response to the Ca2+ antagonist, Flunarizine, suggested that these movements were calcium-dependent. We propose several explanations of the physiological role of these mechanisms and discuss the possibility that fast tilting of the cuticular plate is a physiological movement involving the hair cells at the periphery of the vestibular receptors. The regulation of the vestibular message at the apex of type I hair cells is also considered.

Intracellular free calcium in isolated vestibular hair cells and potassium iontophoresis.

The resting free calcium level was measured in 128 isolated mammalian vestibular sensory cells using the calcium-sensitive dye fura-2. Iontophoresis was used to apply short, localised and limited pulses of K+ which evoked dynamic changes in intracellular free calcium concentration. While most of the type I hair cells tested showed brief reversible and specific calcium responses, some were unresponsive. The changes in intracellular free calcium were also measured by videomicroscopic analysis. Iontophoretic application of K+ ions is shown to be a suitable method for inducing fast, transient changes in intracellular free calcium in vestibular hair cells. This technique could be useful for applying several ions and charged molecules such as amino acids in in-vitro cellular methods.