Sergio Masetto

Position-dependent patterning of spontaneous action potentials in immature cochlear inner hair cells

Spontaneous action potential activity is crucial for mammalian sensory system development. In the auditory system, patterned firing activity has been observed in immature spiral ganglion and brain-stem neurons and is likely to depend on cochlear inner hair cell (IHC) action potentials. It remains uncertain whether spiking activity is intrinsic to developing IHCs and whether it shows patterning. We found that action potentials were intrinsically generated by immature IHCs of altricial rodents and that apical IHCs showed bursting activity as opposed to more sustained firing in basal cells. We show that the efferent neurotransmitter acetylcholine fine-tunes the IHCs resting membrane potential (Vm), and as such is crucial for the bursting pattern in apical cells. Endogenous extracellular ATP also contributes to the Vm of apical and basal IHCs by triggering small-conductance Ca2+-activated K+ (SK2) channels. We propose that the difference in firing pattern along the cochlea instructs the tonotopic differentiation of IHCs and auditory pathway.

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.

miR-96 regulates the progression of differentiation in mammalian cochlear inner and outer hair cells

MicroRNAs (miRNAs) are small noncoding RNAs able to regulate a broad range of protein-coding genes involved in many biological processes. miR-96 is a sensory organ-specific miRNA expressed in the mammalian cochlea during development. Mutations in miR-96 cause nonsyndromic progressive hearing loss in humans and mice. The mouse mutant diminuendo has a single base change in the seed region of the Mir96 gene leading to widespread changes in the expression of many genes. We have used this mutant to explore the role of miR-96 in the maturation of the auditory organ. We found that the physiological development of mutant sensory hair cells is arrested at around the day of birth, before their biophysical differentiation into inner and outer hair cells. Moreover, maturation of the hair cell stereocilia bundle and remodelling of auditory nerve connections within the cochlea fail to occur in miR-96 mutants. We conclude that miR-96 regulates the progression of the physiological and morphological differentiation of cochlear hair cells and, as such, coordinates one of the most distinctive functional refinements of the mammalian auditory system.

Pre- and postsynaptic excitatory action of glutamate agonists on frog vestibular receptors

In order to investigate the localization and the type(s) of excitatory amino acid receptors in the frog vestibular system, the exogenous amino acid agonists Quisqualic acid, Kainic acid and N-methyl-D-aspartic acid were tested on the sensory organ of semicircular canals. Intracellular recordings of the resting discharge from single afferents showed that these agonists exerted a complex excitatory action consisting in a rapid and brief increase in frequency of both EPSPs and spikes, followed by a slower and longer lasting membrane depolarization. The progressive impairment of natural transmitter release achieved by adding Mg2+ or Co2+ in the bath caused a dose-dependent decrease of the agonist-induced afferent discharge, without substantially affecting axonal depolarization. These results suggest that the exogenous amino acid agonists act both pre- and postsynaptically on the vestibular organs. Quisqualic acid and kainic acid were much more potent than N-methyl-D-aspartic acid in inducing excitatory effects, suggesting that the amino acid receptors located on both hair cells and afferent endings are mainly of the non-NMDA type. The present findings, while not excluding that an excitatory amino acid may be the afferent transmitter, highlight its possible function as a presynaptic modulator of the afferent transmission in the frog vestibular system.

Eps8 regulates hair bundle length and functional maturation of mammalian auditory hair cells.

Hair cells of the mammalian cochlea are specialized for the dynamic coding of sound stimuli. The transduction of sound waves into electrical signals depends upon mechanosensitive hair bundles that project from the cells apical surface. Each stereocilium within a hair bundle is composed of uniformly polarized and tightly packed actin filaments. Several stereociliary proteins have been shown to be associated with hair bundle development and function and are known to cause deafness in mice and humans when mutated. The growth of the stereociliar actin core is dynamically regulated at the actin filament barbed ends in the stereociliary tip. We show that Eps8, a protein with actin binding, bundling, and barbed-end capping activities in other systems, is a novel component of the hair bundle. Eps8 is localized predominantly at the tip of the stereocilia and is essential for their normal elongation and function. Moreover, we have found that Eps8 knockout mice are profoundly deaf and that IHCs, but not OHCs, fail to mature into fully functional sensory receptors. We propose that Eps8 directly regulates stereocilia growth in hair cells and also plays a crucial role in the physiological maturation of mammalian cochlear IHCs. Together, our results indicate that Eps8 is critical in coordinating the development and functionality of mammalian auditory hair cells.

Progressive hearing loss and gradual deterioration of sensory hair bundles in the ears of mice lacking the actin-binding protein Eps8L2

Mechanotransduction in the mammalian auditory system depends on mechanosensitive channels in the hair bundles that project from the apical surface of the sensory hair cells. Individual stereocilia within each bundle contain a core of tightly packed actin filaments, whose length is dynamically regulated during development and in the adult. We show that the actin-binding protein epidermal growth factor receptor pathway substrate 8 (Eps8)L2, a member of the Eps8-like protein family, is a newly identified hair bundle protein that is localized at the tips of stereocilia of both cochlear and vestibular hair cells. It has a spatiotemporal expression pattern that complements that of Eps8. In the cochlea, whereas Eps8 is essential for the initial elongation of stereocilia, Eps8L2 is required for their maintenance in adult hair cells. In the absence of both proteins, the ordered staircase structure of the hair bundle in the cochlea decays. In contrast to the early profound hearing loss associated with an absence of Eps8, Eps8L2 null-mutant mice exhibit a late-onset, progressive hearing loss that is directly linked to a gradual deterioration in hair bundle morphology. We conclude that Eps8L2 is required for the long-term maintenance of the staircase structure and mechanosensory function of auditory hair bundles. It complements the developmental role of Eps8 and is a candidate gene for progressive age-related hearing loss.

Ionic currents in regenerating avian vestibular hair cells.

By applying the conventional whole‐cell patch‐clamp technique in combination with the slice procedure, we have investigated the properties of avian semicircular canal hair cells in situ. Passive and active electrical properties of hair cells from control animals have been compared with those of regenerating hair cells following streptomycin treatment (that killed almost all hair cells). Regenerating type II hair cells showed patterns of responses qualitatively similar to those of normal hair cells. However, parameters reflecting the total number of ionic channels and the surface area of type II hair cells changed during recovery—suggesting that new hair cells came from smaller precursors which (with time) reacquired the same electrophysiological properties as normal hair cells. Finally, we have investigated the ionic properties of a small sample of type I hair cells. Ionic currents of regenerating type I hair cells did not show, at least in the temporal window considered (up to 10 weeks from the end of the streptomycin treatment), the typical ionic currents of normal type I hair cells, but expressed instead ionic currents resembling those of type II hair cells. The possibility that regenerating type I hair cells can transdifferentiate from type II hair cells is therefore suggested.

Burst activity and ultrafast activation kinetics of CaV1.3 Ca2+ channels support presynaptic activity in adult gerbil hair cell ribbon synapses

•  The transfer of sound information to the brain relies on the precise release of neurotransmitter from sensory inner hair cell (IHC) ribbon synapses. •  Neurotransmitter release from IHCs is triggered by Ca2+ entry mainly through one type of Ca2+ channel (CaV1.3). •  In this study we show that in near‐physiological conditions Ca2+ channels open very rapidly following a stimulus with a delay of about 50 μs. •  Despite the open probability of the Ca2+ channels being very low, they can switch to a burst‐like mode during a stimulus, maximizing Ca2+ influx into IHCs. •  These results help us to better understand how IHCs are able to accomplish high‐fidelity signal transfer at individual auditory ribbon synapses.

Intercellular K+ accumulation depolarizes Type I vestibular hair cells and their associated afferent nerve calyx

Mammalian vestibular organs contain two types of sensory receptors, named Type I and Type II hair cells. While Type II hair cells are contacted by several small afferent nerve terminals, the basolateral surface of Type I hair cells is almost entirely enveloped by a single large afferent nerve terminal, called calyx. Moreover Type I, but not Type II hair cells, express a low-voltage-activated outward K(+) current, I(K,L), which is responsible for their much lower input resistance (Rm) at rest as compared to Type II hair cells. The functional meaning of I(K,L) and associated calyx is still enigmatic. By combining the patch-clamp whole-cell technique with the mouse whole crista preparation, we have recorded the current- and voltage responses of in situ hair cells. Outward K(+) current activation resulted in K(+) accumulation around Type I hair cells, since it induced a rightward shift of the K(+) reversal potential the magnitude of which depended on the amplitude and duration of K(+) current flow. Since this phenomenon was never observed for Type II hair cells, we ascribed it to the presence of a residual calyx limiting K(+) efflux from the synaptic cleft. Intercellular K(+) accumulation added a slow (τ>100ms) depolarizing component to the cell voltage response. In a few cases we were able to record from the calyx and found evidence for intercellular K(+) accumulation as well. The resulting depolarization could trigger a discharge of action potentials in the afferent nerve fiber. Present results support a model where pre- and postsynaptic depolarization produced by intercellular K(+) accumulation cooperates with neurotransmitter exocytosis in sustaining afferent transmission arising from Type I hair cells. While vesicular transmission together with the low Rm of Type I hair cells appears best suited for signaling fast head movements, depolarization produced by intercellular K(+) accumulation could enhance signal transmission during slow head movements.

Calcium channels functional roles in the frog semicircular canal

Different types of voltage-operated calcium channels have been described in hair cells; however, no clear functional role has been assigned to them. As a first functional characterization of vestibular calcium channels, we studied the effect of several calcium channel agonists and antagonists on whole nerve firing rate in an isolated frog semicircular canal preparation. Resting activity was affected by all dihydropyridines tested and by ω-conotoxin GVIA, whereas only nimodipine was able to reduce the mechanically evoked activity. These results indicate that nimodipine-sensitive channels play a major role in afferent transmitter release, and ω-conotoxin GVIA sensitive channels regulate the afferent firing (possibly on the postsynaptic side) but with a less important role.