Immature Inner Hair Cells Research Articles

Functional and developmental changes in the inner hair cell ribbon synapses caused by Myosin VI knockout and deafness-inducing point mutation

Hearing loss is one of the most common neurosensory disorders in humans, and above half of hearing loss is caused by gene mutations. Among more than 100 genes that cause non-syndromic hearing loss, myosin VI (MYO6) is typical in terms of the complexity of underlying mechanisms, which are not well understood. In this study, we used both knock-out (Myo6−/−) and point mutation (Myo6C442Y) mice as animal models, performed whole-cell patch-clamp recording and capacitance measurement in the inner hair cells (IHCs) in the cochlea, and sought to reveal potential functional and developmental changes in their ribbon synapses. In Myo6−/− cochleae of both before (P8-10) and after hearing onset (P18-20), exocytosis from IHCs, measured in whole-cell capacitance change (ΔCm), was significantly reduced, Ca2+ current amplitude (ICa) was unchanged, but Ca2+ voltage dependency was differently altered, causing significant increase in Ca2+ influx in mature IHCs but not in immature IHCs. In immature IHCs of Myo6C442Y/C442Y cochleae, neither ΔCm nor ICa was altered, but both were reduced in mature IHCs of the same animal model. Furthermore, while the reduction of exocytosis was caused by a combination of the slower rate of depleting readily releasable (RRP) pool of synaptic vesicles and slower sustained release rate (SRR) in Myo6−/− immature IHCs, it was likely due to smaller RRP and slower SRR in mature IHCs of both animal models. These results expand our understanding of the mechanisms of deafness caused by MYO6 mutations, and provide a solid theoretical and scientific basis for the diagnosis and treatment of deafness.

Distribution and Functional Characteristics of Voltage-Gated Sodium Channels in Immature Cochlear Hair Cells.

Voltage-gated sodium channels (VGSCs) are transiently expressed in cochlear hair cells before hearing onset and play an indispensable role in shaping spontaneous activity. In this study, we showed that Na+ currents shaped the spontaneous action potentials in developing mouse inner hair cells (IHCs) by decreasing the time required for the membrane potential to reach the action-potential threshold. In immature IHCs, we identified 9 known VGSC subtypes (Nav1.1α-1.9α), among which Nav1.7α was the most highly expressed subtype and the main contributor to Na+ currents in developing hair cells. Electrophysiological recordings of two cochlea-specific Nav1.7 variants (CbmNav1.7a and CbmNav1.7b) revealed a novel loss-of-function mutation (C934R) at the extracellular linker between segments 5 and 6 of domain II. In addition, post-transcriptional modification events, such as alternative splicing and RNA editing, amended the gating properties and kinetic features of CbmNav1.7a(C934). These results provide molecular and functional characteristics of VGSCs in mammalian IHCs and their contributions to spontaneous physiological activity during cochlear maturation.

Gαi Proteins are Indispensable for Hearing

Background/Aims: From invertebrates to mammals, Gα_i proteins act together with their common binding partner Gpsm2 to govern cell polarization and planar organization in virtually any polarized cell. Recently, we demonstrated that Gα_i3-deficiency in pre-hearing murine cochleae pointed to a role of Gα_i3 for asymmetric migration of the kinocilium as well as the orientation and shape of the stereociliary (“hair”) bundle, a requirement for the progression of mature hearing. We found that the lack of Gα_i3 impairs stereociliary elongation and hair bundle shape in high-frequency cochlear regions, linked to elevated hearing thresholds for high-frequency sound. How these morphological defects translate into hearing phenotypes is not clear. Methods: Here, we studied global and conditional Gnai3 and Gnai2 mouse mutants deficient for either one or both Gα_i proteins. Comparative analyses of global versus Foxg1-driven conditional mutants that mainly delete in the inner ear and telencephalon in combination with functional tests were applied to dissect essential and redundant functions of different Gα_i isoforms and to assign specific defects to outer or inner hair cells, the auditory nerve, satellite cells or central auditory neurons. Results: Here we report that lack of Gα_i3 but not of the ubiquitously expressed Gα_i2 elevates hearing threshold, accompanied by impaired hair bundle elongation and shape in high-frequency cochlear regions. During the crucial reprogramming of the immature inner hair cell (IHC) synapse into a functional sensory synapse of the mature IHC deficiency for Gα_i2 or Gα_i3 had no impact. In contrast, double-deficiency for Gα_i2 and Gα_i3 isoforms results in abnormalities along the entire tonotopic axis including profound deafness associated with stereocilia defects. In these mice, postnatal IHC synapse maturation is also impaired. In addition, the analysis of conditional versus global Gα_i3-deficient mice revealed that the amplitude of ABR wave IV was disproportionally elevated in comparison to ABR wave I indicating that Gα_i3 is selectively involved in generation of neural gain during auditory processing. Conclusion: We propose a so far unrecognized complexity of isoform-specific and overlapping Gα_i protein functions particular during final differentiation processes.

Calcium-Induced Calcium Release during Action Potential Firing in Developing Inner Hair Cells

In the mature auditory system, inner hair cells (IHCs) convert sound-induced vibrations into electrical signals that are relayed to the central nervous system via auditory afferents. Before the cochlea can respond to normal sound levels, developing IHCs fire calcium-based action potentials that disappear close to the onset of hearing. Action potential firing triggers transmitter release from the immature IHC that in turn generates experience-independent firing in auditory neurons. These early signaling events are thought to be essential for the organization and development of the auditory system and hair cells. A critical component of the action potential is the rise in intracellular calcium that activates both small conductance potassium channels essential during membrane repolarization, and triggers transmitter release from the cell. Whether this calcium signal is generated by calcium influx or requires calcium-induced calcium release (CICR) is not yet known. IHCs can generate CICR, but to date its physiological role has remained unclear. Here, we used high and low concentrations of ryanodine to block or enhance CICR to determine whether calcium release from intracellular stores affected action potential waveform, interspike interval, or changes in membrane capacitance during development of mouse IHCs. Blocking CICR resulted in mixed action potential waveforms with both brief and prolonged oscillations in membrane potential and intracellular calcium. This mixed behavior is captured well by our mathematical model of IHC electrical activity. We perform two-parameter bifurcation analysis of the model that predicts the dependence of IHCs firing patterns on the level of activation of two parameters, the SK2 channels activation and CICR rate. Our data show that CICR forms an important component of the calcium signal that shapes action potentials and regulates firing patterns, but is not involved directly in triggering exocytosis. These data provide important insights into the calcium signaling mechanisms involved in early developmental processes.

Otoferlin Couples to Clathrin-Mediated Endocytosis in Mature Cochlear Inner Hair Cells

The encoding of auditory information with indefatigable precision requires efficient resupply of vesicles at inner hair cell (IHC) ribbon synapses. Otoferlin, a transmembrane protein responsible for deafness in DFNB9 families, has been postulated to act as a calcium sensor for exocytosis as well as to be involved in rapid vesicle replenishment of IHCs. However, the molecular basis of vesicle recycling in IHCs is largely unknown. In the present study, we used high-resolution liquid chromatography coupled with mass spectrometry to copurify otoferlin interaction partners in the mammalian cochlea. We identified multiple subunits of the adaptor protein complex AP-2 (CLAP), an essential component of clathrin-mediated endocytosis, as binding partners of otoferlin in rats and mice. The interaction between otoferlin and AP-2 was confirmed by coimmunoprecipitation. We also found that AP-2 interacts with myosin VI, another otoferlin binding partner important for clathrin-mediated endocytosis (CME). The expression of AP-2 in IHCs was verified by reverse transcription PCR. Confocal microscopy experiments revealed that the expression of AP-2 and its colocalization with otoferlin is confined to mature IHCs. When CME was inhibited by blocking dynamin action, real-time changes in membrane capacitance showed impaired synaptic vesicle replenishment in mature but not immature IHCs. We suggest that an otoferlin-AP-2 interaction drives Ca(2+)- and stimulus-dependent compensating CME in mature IHCs.

Developmental alterations in the biophysical properties of Cav1.3 Ca2+ channels in mouse inner hair cells

Prior to hearing onset, spontaneous action potentials activate voltage-gated Cav1.3 Ca2+ channels in mouse inner hair cells (IHCs), which triggers exocytosis of glutamate and excitation of afferent neurons. In mature IHCs, Cav1.3 channels open in response to evoked receptor potentials, causing graded changes in exocytosis required for accurate sound transmission. Developmental alterations in Cav1.3 properties may support distinct roles of Cav1.3 in IHCs in immature and mature IHCs, and have been reported in various species. It is not known whether such changes in Cav1.3 properties occur in mouse IHCs, but this knowledge is necessary for understanding the roles of Cav1.3 in developing and mature IHCs. Here, we describe age-dependent differences in the biophysical properties of Cav1.3 channels in mouse IHCs. In mature IHCs, Cav1.3 channels activate more rapidly and exhibit greater Ca2+-dependent inactivation (CDI) than in immature IHCs. Consistent with the properties of Cav1.3 channels in heterologous expression systems, CDI in mature IHCs is not affected by increasing intracellular Ca2+ buffering strength. However, CDI in immature IHCs is significantly reduced by strong intracellular Ca2+ buffering, which both slows the onset of, and accelerates recovery from, inactivation. These results signify a developmental decline in the sensitivity of CDI to global elevations in Ca2+, which restricts negative feedback regulation of Cav1.3 channels to incoming Ca2+ ions in mature IHCs. Together with faster Cav1.3 activation kinetics, increased reliance of Cav1.3 CDI on local Ca2+ may sharpen presynaptic Ca2+ signals and improve temporal aspects of sound coding in mature IHCs.

Modulation of Cav1.3 Ca2+ channel gating by Rab3 interacting molecule

Neurotransmitter release and spontaneous action potentials during cochlear inner hair cell (IHC) development depend on the activity of Cav1.3 voltage-gated L-type Ca2+ channels. Their voltage- and Ca2+-dependent inactivation kinetics are slower than in other tissues but the underlying molecular mechanisms are not yet understood. We found that Rab3-interacting molecule-2α (RIM2α) mRNA is expressed in immature cochlear IHCs and the protein co-localizes with Cav1.3 in the same presynaptic compartment of IHCs. Expression of RIM proteins in tsA-201 cells revealed binding to the β-subunit of the channel complex and RIM-induced slowing of both Ca2+- and voltage-dependent inactivation of Cav1.3 channels. By inhibiting inactivation, RIM induced a non-inactivating current component typical for IHC Cav1.3 currents which should allow these channels to carry a substantial window current during prolonged depolarizations. These data suggest that RIM2 contributes to the stabilization of Cav1.3 gating kinetics in immature IHCs.

Effects of Kir2.1 gene transfection in cochlear hair cells and application of neurotrophic factors on survival and neurite growth of co-cultured cochlear spiral ganglion neurons

Immature inner hair cells (IHCs) produce spontaneous action potentials, which may be associated with the survival of spiral ganglion neurons (SGNs) during early development. Later, this activity ceases in part by the expression of Kir channels. In the present study, SGNs were co-cultured with organ of Corti in which a Kir2.1 channel was over-expressed in an attempt to block the spontaneous activity of IHCs. The over-expression led to a reduced survival and neurite growth accompanied by increased SGN apoptosis. The enhanced activation of apoptosis was consistent with the inhibition of the survival-promoting pathway and the disruption of [Ca 2+] i homeostasis. Furthermore, the effect of Kir2.1 over-expression can be reversed by exogenous neurotrophic factors (NTFs). These results are consistent with the hypothesis that the earlier-than-normal expression of Kir2.1 in HCs inhibits their spontaneous activity required for SGN survival and neurite growth.

Elementary properties of CaV1.3 Ca2+ channels expressed in mouse cochlear inner hair cells

Mammalian cochlear inner hair cells (IHCs) are specialized to process developmental signals during immature stages and sound stimuli in adult animals. These signals are conveyed onto auditory afferent nerve fibres. Neurotransmitter release at IHC ribbon synapses is controlled by L-type CaV1.3 Ca2+ channels, the biophysics of which are still unknown in native mammalian cells. We have investigated the localization and elementary properties of Ca2+ channels in immature mouse IHCs under near-physiological recording conditions. CaV1.3 Ca2+ channels at the cell pre-synaptic site co-localize with about half of the total number of ribbons present in immature IHCs. These channels activated at about −70 mV, showed a relatively short first latency and weak inactivation, which would allow IHCs to generate and accurately encode spontaneous Ca2+ action potential activity characteristic of these immature cells. The CaV1.3 Ca2+ channels showed a very low open probability (about 0.15 at −20 mV: near the peak of an action potential). Comparison of elementary and macroscopic Ca2+ currents indicated that very few Ca2+ channels are associated with each docked vesicle at IHC ribbon synapses. Finally, we found that the open probability of Ca2+ channels, but not their opening time, was voltage dependent. This finding provides a possible correlation between presynaptic Ca2+ channel properties and the characteristic frequency/amplitude of EPSCs in auditory afferent fibres.

Myosin VI is required for the proper maturation and function of inner hair cell ribbon synapses

The ribbon synapses of auditory inner hair cells (IHCs) undergo morphological and electrophysiological transitions during cochlear development. Here we report that myosin VI (Myo6), an actin-based motor protein involved in genetic forms of deafness, is necessary for some of these changes to occur. By using post-embedding immunogold electron microscopy, we showed that Myo6 is present at the IHC synaptic active zone. In Snell's waltzer mutant mice, which lack Myo6, IHC ionic currents and ribbon synapse maturation proceeded normally until at least post-natal day 6. In adult mutant mice, however, the IHCs displayed immature potassium currents and still fired action potentials, as normally only observed in immature IHCs. In addition, the number of ribbons per IHC was reduced by 30%, and 30% of the remaining ribbons were morphologically immature. Ca2+-dependent exocytosis probed by capacitance measurement was markedly reduced despite normal Ca2+ currents and the large proportion of morphologically mature synapses, which suggests additional defects, such as loose Ca2+-exocytosis coupling or inefficient vesicular supply. Finally, we provide evidence that Myo6 and otoferlin, a putative Ca2+ sensor of synaptic exocytosis also involved in a genetic form of deafness, interact at the IHC ribbon synapse, and we suggest that this interaction is involved in the recycling of synaptic vesicles. Our findings thus uncover essential roles for Myo6 at the IHC ribbon synapse, in addition to that proposed in membrane turnover and anchoring at the apical surface of the hair cells.

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; tau = 79 ms) and a slowly releasable pool (tau = 870 ms). The RRP size and maximal release rate were estimated at approximately 1200 vesicles and approximately 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.

Gerbils can tune in

Gerbils can tune in

Probing the Mechanism of Exocytosis at the Hair Cell Ribbon Synapse

Hearing relies on faithful synaptic transmission at the ribbon synapse of cochlear inner hair cells (IHCs). Postsynaptic recordings from this synapse in prehearing animals had delivered strong indications for synchronized release of several vesicles. The underlying mechanism, however, remains unclear. Here, we used presynaptic membrane capacitance measurements to test whether IHCs release vesicles in a statistically independent or dependent (coordinated) manner. Exocytic changes of membrane capacitance (deltaC(m)) were repeatedly stimulated in IHCs of prehearing and hearing mice by short depolarizations to preferentially recruit the readily releasable pool of synaptic vesicles. A compound Poisson model was devised to describe hair cell exocytosis and to test the analysis. From the trial-to-trial fluctuations of the deltaC(m) we were able to estimate the apparent size of the elementary fusion event (C(app)) at the hair cell synapse to be 96-223 aF in immature and 55-149 aF in mature IHCs. We also approximated the single vesicle capacitance in IHCs by measurements of synaptic vesicle diameters in electron micrographs. The results (immature, 48 aF; mature, 45 aF) were lower than the respective C(app) estimates. This indicates that coordinated exocytosis of synaptic vesicles occurs at both immature and mature hair cell synapses. Approximately 35% of the release events in mature IHCs and approximately 50% in immature IHCs were predicted to involve coordinated fusion, when assuming a geometric distribution of elementary sizes. In summary, our presynaptic measurements indicate coordinated exocytosis but argue for a lesser degree of coordination than suggested by postsynaptic recordings.

Genetic deletion of SK2 channels in mouse inner hair cells prevents the developmental linearization in the Ca2+ dependence of exocytosis

Inner hair cells (IHCs), the primary sensory receptors of the mammalian cochlea, fire spontaneous Ca(2+) action potentials (APs) only before the onset of hearing. Although a role for APs in the developing auditory system has not been determined it could, by analogy with other sensory systems, guide the functional maturation of the cochlea before experience-driven activity begins. Spontaneous APs in immature IHCs are shaped by a variety of ion channels including that of the small conductance Ca(2+)-activated K(+) current (SK2), which is only transiently expressed in immature cells. Using SK2 knockout mice we found that SK2 channels are not required for generating APs but are essential for sustaining continuous repetitive spontaneous AP activity in pre-hearing IHCs. Therefore we used this mutant mouse as a model to study possible developmental implications of disrupted AP activity. Immature mutant IHCs showed impaired exocytotic responses, which are likely to be due to the expression of fewer Ca(2+) channels. Exocytosis was also impaired in adult mutant IHCs, although in this case it resulted from a reduced Ca(2+) efficiency and increased Ca(2+) dependence of the synaptic machinery. Since SK2 channels can only have a functional influence on IHCs during immature development and are not directly involved in neurotransmitter release, the altered Ca(2+) dependence of exocytosis in adult IHCs is likely to be a consequence of their disrupted AP activity at immature stages.

The Presynaptic Function of Mouse Cochlear Inner Hair Cells during Development of Hearing

Before mice start to hear at approximately postnatal day 10, their cochlear inner hair cells (IHCs) spontaneously generate Ca(2+) action potentials. Therefore, immature IHCs could stimulate the auditory pathway, provided that they were already competent for transmitter release. Here, we combined patch-clamp capacitance measurements and fluorimetric [Ca(2+)](i) recordings to study the presynaptic function of IHCs during cochlear maturation. Ca(2+)-dependent exocytosis and subsequent endocytic membrane retrieval were already observed near the date of birth. Ca(2+) action potentials triggered exocytosis in immature IHCs, which probably activates the auditory pathway before it becomes responsive to sound. IHCs underwent profound changes in Ca(2+)-channel expression and secretion during their postnatal development. Ca(2+)-channel expression increased toward the end of the first week, providing for large secretory responses during this period and thereafter declined to reach mature levels. The efficacy whereby Ca(2+) influx triggers exocytosis increased toward maturation, such that vesicle fusion caused by a given Ca(2+) current occurred faster in mature IHCs. The observed changes in Ca(2+)-channel expression and synaptic efficacy probably reflected the ongoing synaptogenesis in IHCs that had been described previously in morphological studies.