J. Lehouelleur

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), α‐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‐dinitro‐quinoxaline‐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.

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

Abstract 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.

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.

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.

Electrophysiological properties of the utricular primary transducer are modified during development under hypergravity

The electrophysiological development of hair cells between birth and the eight postnatal day (P8) was studied in the utricular macula of rats gestated in nest boxes mounted upon a centrifuge, subjecting the animals to a gravitational force of 2G. Whole‐cell voltage‐clamp recordings were made on cells in the acutely isolated epithelium. Cells were accessed through a tear in the epithelium, no enzymatic dissociation procedures were employed. Under artificially enhanced gravity, the whole cell conductance was dramatically altered in the two types of hair cells. Significant increases occurred from P3–4 in the type I cells while in the type II cells, the effect was delayed until P7–8. Fourfold and threefold increases of the mean slope conductance were observed at P7–8 in the type I and type II hair cells, respectively. These results indicate that the electrophysiological properties of a primary transducer such as utricle may be modified by variation of the primary stimulus during development.

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 μm 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 ω‐agatoxin IVA (1 μm), a specific P‐ and Q‐type blocker, nor ω‐conotoxin GVIA (1 μm), 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.

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.

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.

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°, 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°, 200 ms, 0.5Hz) evoked increases of [Ca2+]ifrom 50±23 nM to 139±79 (n=12). In the opposite direction, the mechanical stimulations (8°, 400ms, 0.5Hz) 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 μM 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.

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.