Didier Dulon

Potassium-depolarization induces motility in isolated outer hair cells by an osmotic mechanism

Outer hair cells in vitro contract in response to various stimuli: electrical stimulation, K+-depolarization, elevation of intracellular calcium or osmotic changes of the extracellular medium. The characteristics of motile responses induced by K+-depolarization, osmotic changes, and calcium injection were compared in this study in order to delineate the underlying mechanisms. Slow shape changes in outer hair cells were induced by changes of the osmolality or the K+/Na+-ratio of the bathing medium, or by intracellular injections of calcium. K+- and osmotically induced contractions of isolated outer hair cells had identical morphological features and the same rate (50-200 nm/s) and amplitude (up to greater than 10% of original length) of shortening. The shortening of the cells was linearly related to an increase in volume in both cases. In contrast, the active contraction induced by Ca2+/ATP exhibited a somewhat faster rate and no increase in volume. Furthermore, the K+-induced contractions in outer hair cells, unlike those reported in smooth muscle cells, were unaffected by the removal of external Ca2+ (i.e. medium without Ca2+/Mg2+ and supplemented with 1 mM EGTA) or the presence of D600, an inhibitor of the Ca2+ inward current. The results strongly suggest that K+ induces shape changes of outer hair cells via osmotic forces and that intracellular calcium mediates contractions by a different mechanism.

Otoferlin Is Critical for a Highly Sensitive and Linear Calcium-Dependent Exocytosis at Vestibular Hair Cell Ribbon Synapses

Otoferlin, a C2-domain-containing Ca2+ binding protein, is required for synaptic exocytosis in auditory hair cells. However, its exact role remains essentially unknown. Intriguingly enough, no balance defect has been observed in otoferlin-deficient (Otof−/−) mice. Here, we show that the vestibular nerve compound action potentials evoked during transient linear acceleration ramps in Otof−/− mice display higher threshold, lower amplitude, and increased latency compared with wild-type mice. Using patch-clamp capacitance measurement in intact utricles, we show that type I and type II hair cells display a remarkable linear transfer function between Ca2+ entry, flowing through voltage-activated Ca2+ channels, and exocytosis. This linear Ca2+ dependence was observed when changing the Ca2+ channel open probability or the Ca2+ flux per channel during various test potentials. In Otof−/− hair cells, exocytosis displays slower kinetics, reduced Ca2+ sensitivity, and nonlinear Ca2+ dependence, despite morphologically normal synapses and normal Ca2+ currents. We conclude that otoferlin is essential for a high-affinity Ca2+ sensor function that allows efficient and linear encoding of low-intensity stimuli at the vestibular hair cell synapse.

Control of Exocytosis by Synaptotagmins and Otoferlin in Auditory Hair Cells

In pre-hearing mice, vesicle exocytosis at cochlear inner hair cell (IHC) ribbon synapses is triggered by spontaneous Ca2+ spikes. At the onset of hearing, IHC exocytosis is then exclusively driven by graded potentials, and is characterized by higher Ca2+ efficiency and improved synchronization of vesicular release. The molecular players involved in this transition are still unknown. Here we addressed the involvement of synaptotagmins and otoferlin as putative Ca2+ sensors in IHC exocytosis during postnatal maturation of the cochlea. Using cell capacitance measurements, we showed that Ca2+-evoked exocytosis in mouse IHCs switches from an otoferlin-independent to an otoferlin-dependent mechanism at postnatal day 4. During this early exocytotic period, several synaptotagmins (Syts), including Syt1, Syt2 and Syt7, were detected in IHCs. The exocytotic response as well as the release of the readily releasable vesicle pool (RRP) was, however, unchanged in newborn mutant mice lacking Syt1, Syt2 or Syt7 (Syt1−/−, Syt2−/− and Syt7−/− mice). We only found a defect in RRP recovery in Syt1−/− mice which was apparent as a strongly reduced response to repetitive stimulations. In post-hearing Syt2−/− and Syt7−/− mutant mice, IHC synaptic exocytosis was unaffected. The transient expression of Syt1 and Syt2, which were no longer detected in IHCs after the onset of hearing, indicates that these two most common Ca2+-sensors in CNS synapses are not involved in mature IHCs. We suggest that otoferlin underlies highly efficient Ca2+-dependent membrane-membrane fusion, a process likely essential to increase the probability and synchrony of vesicle fusion events at the mature IHC ribbon synapse.

Localization and developmental expression of BK channels in mammalian cochlear hair cells.

The expression of Slo channels (alpha subunits of BK channels) was investigated in the developing mouse cochlea using a polyclonal antibody against the C-terminal part of the protein (residues 1098-1196). The first BK channel immunoreactivity was observed in the cochlea at E18, where it was localized within the cytoplasm of cells lining the area of the organ of Corti and the spiral ganglion. There was an increase of immunoreactivity in all cells bordering the scala media (supporting and hair cells of the organ of Corti, the stria vascularis and the Reissners membrane) in the following stages (postnatal day [P] 0 and P6). From P12 to adult, a strong membranous labeling, increasing with age, appeared in inner hair cells. The distribution of BK channels was mainly observed as dense elongated plaques localized in the lateral membrane below the cuticular plate. In addition, a more discrete immunolabeling for BK channels, as punctuated dots, was observed in the synaptic area of inner hair cells. This dual localization of BK channels within inner hair cells was confirmed by a different technique using a fluorescently labeled high-affinity ligand of these channels: IbTX-D19C-Alexa488. We demonstrated under patch clamp experiments that this fluorescent toxin conserved its native property, i.e. to reversibly inhibit BK currents in isolated inner hair cells. The fluorescent toxin, both in living or fixed tissues, also showed a preferential binding to mature inner hair cells with a similar subcellular distribution described above using immunocytochemical technique. Overall, our present results confirm the appearance of membranous BK channels around P12 in mouse inner hair cells, an age at which the auditory system becomes functional. The expression of BK channels in mature inner hair cells, near the site of mechanical-transduction, might serve to limit receptor potential attenuation due to the space constant, and thus permitting these sensory cells to function as fast and sensitive transducers.

Osmotically induced motility of outer hair cells: implications for Menière's disease

SummaryOsmolarity changes in inner ear fluids have long been considered to be contributing factors to Menières disease. Our present study demonstrates that small changes in the osmolarity of a surrounding in vitro medium induce fast contractions (hypo-osmotic solution) or elongations (hyperosmotic solution) in isolated outer hair cells of the guinea pig. These changes were reversible upon returning cells to iso-osmotic conditions. Up to five cycles of shape change could be sustained by these cells without obvious detriment to their morphology. These findings suggest that fluctuant changes in osmolarity of inner ear fluids can result in similar fluctuant changes of hair cell shape. Since the outer hair cells may control cochlear micromechanics and function by their active motility, osmotically induced abnormalities of cell dimensions and motility may contribute to the audiological manifestations of fluctuant hearing loss.

Extracellular ATP elevates cytosolic Ca2+ in cochlear inner hair cells.

The local extracellular application of ATP to isolated sensory inner hair cells (IHC) generated a rapid and transient increase in the concentration of cytosolic free Ca2+, peaking within 1 to 5 s. The dose-response curve indicated a half-max stimulation to be 5 microM ATP. The application of the structural derivatives ADP and alpha-beta-methyleneATP did not generate significant Ca2+ response. By contrast, ATP-alpha-S, an agonist for P2z receptor, was fully active in generating Ca2+ responses. In the absence of extracellular free calcium, the ATP-induced Ca2+ increase was still observed, indicating that ATP generated the liberation of Ca2+ from internal stores. These results suggest that extracellular ATP, through an activation of P2-purinergic receptors, may have a neuromodulatory role in the cochlear physiology at the level of the IHC.

Calcium-and Otoferlin-Dependent Exocytosis by Immature Outer Hair Cells

Immature cochlear outer hair cells (OHCs) make transient synaptic contacts (ribbon synapses) with type I afferent nerve fibers, but direct evidence of synaptic vesicle exocytosis is still missing. We thus investigated calcium-dependent exocytosis in murine OHCs at postnatal day 2 (P2)–P3, a developmental stage when calcium current maximum amplitude was the highest. By using time-resolved patch-clamp capacitance measurements, we show that voltage step activation of L-type calcium channels triggers fast membrane capacitance increase. Capacitance increase displayed two kinetic components, which are likely to reflect two functionally distinct pools of synaptic vesicles, a readily releasable pool (RRP; τ = 79 ms) and a slowly releasable pool (τ = 870 ms). The RRP size and maximal release rate were estimated at ∼1200 vesicles and ∼15,000 vesicles/s, respectively. In addition, we found a linear relationship between capacitance increase and calcium influx, like in mature inner hair cells (IHCs). These results give strong support to the existence of efficient calcium-dependent neurotransmitter release in immature OHCs. Moreover, we show that immature OHCs, just like immature IHCs, are able to produce regenerative calcium-dependent action potentials that could trigger synaptic exocytosis in vivo. Finally, the evoked membrane capacitance increases were abolished in P2–P3 OHCs from mutant Otof−/− mice defective for otoferlin, despite normal calcium currents. We conclude that otoferlin, the putative major calcium sensor at IHC ribbon synapses, is essential to synaptic exocytosis in immature OHCs too.

Comparative uptake of gentamicin, netilmicin, and amikacin in the guinea pig cochlea and vestibule.

The kinetics of the entry of three aminoglycosides into inner-ear tissues of the guinea pig after acute and chronic administration were compared: gentamicin toxic to the cochlea and the vestibule, amikacin preferentially cochleotoxic, and netilmicin of low ototoxic liability. During constant intravenous infusion, levels of the three drugs in plasma tended to reach a plateau after 1 h, while levels in perilymph did not reach a plateau within 6 h. The drug concentrations in both vestibular and cochlear tissues quickly reached saturation. Amikacin and gentamicin concentrations were similar in vestibular and cochlear tissues, while netilmicin values were somewhat lower. After 1 week of chronic treatment (100 mg of drug per kg of body weight daily subcutaneously), levels of gentamicin and amikacin in tissue were similar to each other and were not significantly different between cochlear and vestibular tissues. Netilmicin concentrations again were somewhat lower in the tissues, but identical to those of the other drugs in the perilymph. After 3 weeks of treatment, all of the drugs were equally distributed in the inner-ear tissues. Release of the drug from the tissues after the 3-week treatment was faster for amikacin (83% decrease after 20 days) than for netilmicin and gentamicin (approximately 50% decrease). There was no correlation, under any of the experimental conditions, between the drug concentrations and their degrees of toxicity. These results demonstrate that selective aminoglycoside ototoxicity cannot be explained by a preferential uptake or accumulation of drugs in the afflicted tissues or in the perilymph.

Dynamic changes following combined treatment with gentamicin and ethacrynic acid with and without acoustic stimulation: cellular uptake and functional correlates

Regional selectivity of gentamicin (GM) ototoxicity was studied in guinea pigs (GPs) using electrophysiological, morphological, autoradiographic and immunohistological observations following combined treatment with GM (150 mg/kg i.m.) and ethacrynic acid (EA) (30 mg/kg i.c. or i.v., 1.5 h after GM injection). The GPs were either continuously stimulated every 5 min with a series of 256 clicks (70 dB peSPL, 10/s) during 3 h for monitoring fast changes in VIII nerve compound action potential (CAP) after the EA injection, and thereafter kept in the animal quarters (background noise of 60 dB SPL) (group I), or similarly monitored for only 10 min after the EA injection and thereafter kept in a soundproof room (around 0 dB SPL) (group II). Whenever GM labelling was observed it was localized only in the sensory hair cells. From 3 h after EA injection, the GPs in group I presented threshold elevations in the high-frequency region, which progressed to 60-80 dB at all frequencies at and after 48 h. Parallel to the threshold pattern, GM uptake in outer hair cells (OHCs) was seen with an increasing concentration from apex toward base from 3 to 24 h, while after 48 h almost all OHCs were destroyed and inner hair cells (IHCs) were marked by GM. In group II no changes in CAP thresholds were observed until more than 24 h, although GM was detected in the hair cells from 6 h on. At this early stage, the distribution of GM lacked a clear pattern, particularly without a clear apex-base gradient, and GM deposits were found only around the basal body. However in both groups, in late stage (greater than 24 h), the base-apex gradient was more pronounced and GM was found throughout the cell body, with a marked concentration below the cuticular plate. These results suggest that GM may penetrate hair cells around the basal body and that activating the cells by sound potentiates both GM uptake and its intracellular toxicity.

Cholinergic Responses in Developing Outer Hair Cells of the Rat Cochlea

Acetylcholine‐evoked currents were investigated using the conventional whole‐cell patch‐clamp recording technique in developing outer hair cells (OHCs). The cells were isolated from the rat cochlea at different stages of postnatal development ranging from day 4 (P4) to P30. Acetylcholine‐evoked currents could be recorded at P6 and P8. At this developmental stage, the majority of OHCs displayed inward nicotinic‐like currents near the resting membrane potential. These cholinergic currents zeroed near 0 mV, as expected for a non‐selective cation current, and could be reversibly blocked by d‐tubocurarine. At P12 and adult stage, the cholinergic response of OHCs switched to an outward current reversing near EK and displaying a bell shape peaking between ‐40 and ‐30 mV. This change in polarity of the acetylcholine response during postnatal development might be explained by progressive functional coupling between acetylcholine ionotropic receptors permeable to Ca2+ and nearby Ca2+‐activated K+ channels at the synaptic pole of OHCs.