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.

The resting transducer current drives spontaneous activity in prehearing mammalian cochlear inner hair cells.

Spontaneous Ca(2+)-dependent electrical activity in the immature mammalian cochlea is thought to instruct the formation of the tonotopic map during the differentiation of sensory hair cells and the auditory pathway. This activity occurs in inner hair cells (IHCs) during the first postnatal week, and the pattern differs along the cochlea. During the second postnatal week, which is before the onset of hearing in most rodents, the resting membrane potential for IHCs is apparently more hyperpolarized (approximately -75 mV), and it remains unclear whether spontaneous action potentials continue to occur. We found that when mouse IHC hair bundles were exposed to the estimated in vivo endolymphatic Ca(2+) concentration (0.3 mm) present in the immature cochlea, the increased open probability of the mechanotransducer channels caused the cells to depolarize to around the action potential threshold (approximately -55 mV). We propose that, in vivo, spontaneous Ca(2+) action potentials are intrinsically generated by IHCs up to the onset of hearing and that they are likely to influence the final sensory-independent refinement of the developing cochlea.

New developments in understanding the mechanisms and function of spontaneous electrical activity in the developing mammalian auditory system.

In the mature mammalian auditory system, inner hair cells are responsible for converting sound-evoked vibrations into graded electrical responses, resulting in release of neurotransmitter and neuronal transmission via the VIIIth cranial nerve to auditory centres in the central nervous system. Before the cochlea can reliably respond to sound, inner hair cells are not merely immature quiescent pre-hearing cells, but instead are capable of generating 'spontaneous' calcium-based action potentials. The resulting calcium signal promotes transmitter release that drives action potential firing in developing spiral ganglion neurones. These early signalling events that occur before sound-evoked activity are thought to be important in guiding and refining the initial phases of development of the auditory circuits. This review will summarise our current knowledge of the mechanisms that underlie spontaneous action potentials in developing inner hair cells and how these events are triggered and regulated.

Depolarization of cochlear outer hair cells evokes active hair bundle motion by two mechanisms.

There is current debate about the origin of mechanical amplification whereby outer hair cells generate force to augment the sensitivity and frequency selectivity of the mammalian cochlea. To distinguish contributions to force production from the mechanotransducer (MET) channels and somatic motility, we have measured hair bundle motion during depolarization of individual outer hair cells in isolated rat cochleas. Depolarization evoked rapid positive bundle deflections that were reduced by perfusion with the MET channel blocker dihydrostreptomycin, with no effect on the nonlinear capacitance that is a manifestation of prestin-driven somatic motility. However, the movements were also diminished by Na salicylate and depended on the intracellular anion, properties implying involvement of the prestin motor. Furthermore, depolarization of one outer hair cell caused motion of neighboring hair bundles, indicating overall motion of the reticular lamina. Depolarization of solitary outer hair cells caused cell-length changes whose voltage-activation range depended on the intracellular anion but were insensitive to dihydrostreptomycin. These results imply that both the MET channels and the somatic motor participate in hair bundle motion evoked by depolarization. It is conceivable that the two processes can interact, a signal from the MET channels being capable of modulating the activity of the prestin motor.

The transduction channel filter in auditory hair cells.

In the first step in auditory transduction, sound-induced vibrations of the stereociliary bundles on the sensory hair cells are converted into electrical signals by opening of mechanotransducer channels. Faithful transduction and hence auditory performance will be limited by the kinetic properties of these channels. We have measured the time course of mechanotransducer currents in turtle and rat auditory hair cells during rapid deflections of the hair bundle. Current activation in the turtle had a time constant that decreased 10-fold with stimulus amplitude to a limiting value of approximately 50 micros. Lowering the external Ca2+ concentration slowed both activation and adaptation time constants. Similar effects were seen in hair cells tuned to low and high frequencies, but the overall kinetics was slower in low-frequency cells. In rat outer hair cells, the time courses of both activation and adaptation were at least 10-fold faster. Although activation kinetics was too fast to characterize accurately, the adaptation time constants in the rat, like the turtle, were Ca2+ dependent and faster in hair cells tuned to higher frequencies. The results imply that mechanotransducer channels operate similarly in turtle and rat but are faster in the mammal to accommodate its higher frequency range of hearing. We suggest that the kinetics of channel activation and adaptation imposes a bandpass filter on transduction, with a center frequency matched to the frequencies detected by the hair cell, which may improve the signal-to-noise ratio near threshold.

Development of outward potassium currents in inner and outer hair cells from the embryonic mouse cochlea.

We recorded K(+) currents in inner (IHCs) and outer (OHCs) hair cells from mice at embryonic days 16 and 18 and on the day of birth (PO) to characterize their early physiological differentiation. In both cell types, outward currents increased in size during late embryonic development, in cells situated in both the apical and basal coils of the cochlea. Currents increased up to six-fold, with current density increasing four-fold. Currents in basal cells were generally larger than in the apex, and currents in IHCs were larger than in OHCs at any given stage. In OHCs, they were initially non-inactivating but gained the partial inactivation characteristic of the K(+) current of neonatal mouse cochlear hair cells, I(K,neo), by day 18 in the base and by P0 in the apex. In IHCs, there was little change, other than in amplitude, with partial inactivation already evident in the base by embryonic day 16. These results suggest that changes in the channel complement of OHCs occur within a few days of terminal mitosis, whereas in IHCs any such development would occur earlier. The progressive development of K(+) currents correlates with a developmental delay of around 2 days from the base to the apex of the cochlea.

Fast adaptation of mechanoelectrical transducer channels in mammalian cochlear hair cells.

Outer hair cells are centrally involved in the amplification and frequency tuning of the mammalian cochlea, but evidence about their transducing properties in animals with fully developed hearing is lacking. Here we describe measurements of mechanoelectrical transducer currents in outer hair cells of rats between postnatal days 5 and 18, before and after the onset of hearing. Deflection of hair bundles using a new rapid piezoelectric stimulator evoked transducer currents with ultra-fast activation and adaptation kinetics. Fast adaptation resembled the same process in turtle hair cells, where it is regulated by changes in stereociliary calcium. It is argued that sub-millisecond transducer adaptation can operate in outer hair cells under the ionic, driving force and temperature conditions that prevail in the intact mammalian cochlea.

Fast Ca2+ signals at mouse inner hair cell synapse: a role for Ca2+-induced Ca2+ release.

Inner hair cells of the mammalian cochlea translate acoustic stimuli into 'phase-locked' nerve impulses with frequencies of up to at least 1 kHz. Little is known about the intracellular Ca2+ signal that links transduction to the release of neurotransmitter at the afferent synapse. Here, we use confocal microscopy to provide evidence that Ca2+-induced Ca2+ release (CICR) may contribute to the mechanism. Line scan images (2 ms repetition rate) of neonatal mouse inner hair cells filled with the fluorescent indicator FLUO-3, revealed a transient increase in intracellular Ca2+ concentration ([Ca2+]i) during brief (5-50 ms) depolarizing commands under voltage clamp. The amplitude of the [Ca2+]i transient depended upon the Ca2+ concentration in the bathing medium in the range 0-1.3 mM. [Ca2+]i transients were confined to a region near the plasma membrane at the base of the cell in the vicinity of the afferent synapses. The change in [Ca2+]i appeared uniform throughout the entire basal sub-membrane space and we were unable to observe hotspots of activity. Both the amplitude and the rate of rise of the [Ca2+]i transient was reduced by external ryanodine (20 microM), an agent that blocks Ca2+ release from the endoplasmic reticulum. Intracellular Cs+, commonly used to record at presynaptic sites, produced a similar effect. We conclude that both ryanodine and intracellular Cs+ block CICR in inner hair cells. We discuss the contribution of CICR to the measured [Ca2+]i transient, the implications for synaptic transmission at the afferent synapse and the significance of its sensitivity to intracellular Cs+.