High Frequency Hair Cells Research Articles

The Coupling between Ca2+ Channels and the Exocytotic Ca2+ Sensor at Hair Cell Ribbon Synapses Varies Tonotopically along the Mature Cochlea.

The cochlea processes auditory signals over a wide range of frequencies and intensities. However, the transfer characteristics at hair cell ribbon synapses are still poorly understood at different frequency locations along the cochlea. Using recordings from mature gerbils, we report here a surprisingly strong block of exocytosis by the slow Ca2+ buffer EGTA (10 mM) in basal hair cells tuned to high frequencies (∼30 kHz). In addition, using recordings from gerbil, mouse, and bullfrog auditory organs, we find that the spatial coupling between Ca2+ influx and exocytosis changes from nanodomain in low-frequency tuned hair cells (∼<2 kHz) to progressively more microdomain in high-frequency cells (∼>2 kHz). Hair cell synapses have thus developed remarkable frequency-dependent tuning of exocytosis: accurate low-latency encoding of onset and offset of sound intensity in the cochlea's base and submillisecond encoding of membrane receptor potential fluctuations in the apex for precise phase-locking to sound signals. We also found that synaptic vesicle pool recovery from depletion was sensitive to high concentrations of EGTA, suggesting that intracellular Ca2+ buffers play an important role in vesicle recruitment in both low- and high-frequency hair cells. In conclusion, our results indicate that microdomain coupling is important for exocytosis in high-frequency hair cells, suggesting a novel hypothesis for why these cells are more susceptible to sound-induced damage than low-frequency cells; high-frequency inner hair cells must have a low Ca2+ buffer capacity to sustain exocytosis, thus making them more prone to Ca2+-induced cytotoxicity.SIGNIFICANCE STATEMENT In the inner ear, sensory hair cells signal reception of sound. They do this by converting the sound-induced movement of their hair bundles present at the top of these cells, into an electrical current. This current depolarizes the hair cell and triggers the calcium-induced release of the neurotransmitter glutamate that activates the postsynaptic auditory fibers. The speed and precision of this process enables the brain to perceive the vital components of sound, such as frequency and intensity. We show that the coupling strength between calcium channels and the exocytosis calcium sensor at inner hair cell synapses changes along the mammalian cochlea such that the timing and/or intensity of sound is encoded with high precision.

Hair cell BK channels interact with RACK1, and PKC increases its expression on the cell surface by indirect phosphorylation

Large conductance (BK) calcium activated potassium channels (Slo) are ubiquitous and implicated in a number of human diseases including hypertension and epilepsy. BK channels consist of a pore forming α-subunit (Slo) and a number of accessory subunits. In hair cells of nonmammalian vertebrates these channels play a critical role in electrical resonance, a mechanism of frequency selectivity. Hair cell BK channel clusters on the surface and currents increase along the tonotopic axis and contribute significantly to the responsiveness of these hair cells to sounds of high frequency. In contrast, messenger RNA levels encoding the Slo gene show an opposite decrease in high frequency hair cells. To understand the molecular events underlying this paradox, we used a yeast two-hybrid screen to isolate binding partners of Slo. We identified Rack1 as a Slo binding partner and demonstrate that PKC activation increases Slo surface expression. We also establish that increased Slo recycling of endocytosed Slo is at least partially responsible for the increased surface expression of Slo. Moreover, analysis of several PKC phosphorylation site mutants confirms that the effects of PKC on Slo surface expression are likely indirect. Finally, we show that Slo clusters on the surface of hair cells are also increased by increased PKC activity and may contribute to the increasing amounts of channel clusters on the surface of high-frequency hair cells.

Prestin Surface Expression and Activity Are Augmented by Interaction with MAP1S, a Microtubule-associated Protein

Prestin is a member of the SLC26 family of anion transporters that is responsible for outer hair cell (OHC) electromotility. Measures of voltage-evoked charge density (Q(sp)) of prestin indicated that the protein is highly expressed in OHCs, with single cells expressing up to 10 million molecules within the lateral membrane. In contrast, charge density measures in transfected cells indicated that they express, at best, only a fifth as many proteins on their surface. We sought to determine whether associations with other OHC-specific proteins could account for this difference. Using a yeast two-hybrid technique, we found microtubule-associated protein 1S (MAP1S) bound to prestin. The interaction was limited to the STAS domain of prestin and the region connecting the heavy and light chain of MAP1S. Using reciprocal immunoprecipitation and Forster resonance energy transfer, we confirmed these interactions. Furthermore, co-expression of prestin with MAP1S resulted in a 2.7-fold increase in Q(sp) in single cells that was paralleled by a 2.8-fold increase in protein surface expression, indicating that the interactions are physiological. Quantitative PCR data showed gradients in the expression of prestin and MAP1S across the tonotopic axis that may partially contribute to a previously observed 6-fold increase in Q(sp) in high frequency hair cells. These data highlight the importance of protein partner effects on prestin.

Cochlear dead zones: What are they and how do you detect them?

Cochlear dead zones: What are they and how do you detect them?

The relationship between noise-induced hearing loss and hair cell loss in rats

Noise-induced hearing loss (NIHL) and hair cell loss are known to show only a moderate correlation. One reason for this is that NIHL may reflect not only the sum of dead hair cells, but also the sum of impaired but still living hair cells. This report compares hair cell loss in different cochlear regions in rats with noise-induced compound action potential (CAP) threshold elevation at corresponding frequencies. CAP threshold elevation and hair cell loss were determined 4 weeks after noise exposure. In the apical turn (<35% from the apex) there was no hair cell loss even when a 60 dB CAP threshold elevation was induced. In the region of 40–60% from the apex in the middle turn, significant hair cell loss was not observed until CAP threshold elevation exceeded about 40–50 dB. This critical level decreased towards the basal turn. In the basal turn, outer hair cell (OHC) loss was observed in almost all of the noise-exposed rats, even in some cases without detectable NIHL, but inner hair cell (IHC) loss was still not observed until 50 dB threshold elevation. In the region of 75–90% from the apex related to the highest frequencies tested in this study (30–40 kHz), a linear NIHL/OHC loss relationship was observed. The results of this paper suggest that the high frequency hair cells in rat cochlea may die relatively rapidly after injury, leading to a linear relation between NIHL and hair cell loss, but that the low frequency hair cells may survive without auditory function.

Topological and developmental gradients of calbindin expression in the chick's inner ear.

Mobile intracellular calcium buffers play an important role in regulating calcium flux into mechanosensory hair cells and calbindin D-28k is expressed at high levels in the chick's basilar papilla. We have used RT-PCR, in situ hybridization, and immunohistology to demonstrate that calbindin expression varies systematically according to hair cell position and developmental age. RT-PCR using microdissected quarters of the posthatch basilar papilla showed that mRNA levels were lowest in the (low frequency) apex and higher in basal quadrants. In situ hybridization revealed calbindin mRNA in posthatch hair cells and supporting cells, with more intense labeling of hair cells from basal (high frequency) positions. A similar topology was obtained with calbindin antibodies. Neither calbindin riboprobe nor calbindin antibody labeled cochlear neurons. In contrast, a subset of large vestibular neurons and their calyciform endings onto Type I vestibu lar hair cells were strongly labeled by the calbindin antibody, while vestibular hair cells were negative for calbindin immunoreactivity. Likewise, calbindin in situ hybridization was negative for vestibular hair cells but positive in a subset of larger vestibular neurons. Calbindin mRNA was detected in hair cells of the basal half of the papilla at embryonic day 10 (E10) and calbindin immunoreactivity was detected at E12. Hair cells in the apical half of the papilla had equivalent calbindin expression two days later. Immunoreactivity appeared in abneural supporting cells days later than in hair cells, and not until E20 in neurally located supporting cells. These results demonstrate that calbindin message and protein levels are greater in high-frequency hair cells. This "tonotopic" gradient may result from the stabilization of a basal-to-apical developmental gradient and could be related at least in part to calcium channel expression along this axis.

High-Frequency Auditory Feedback Is Not Required for Adult Song Maintenance in Bengalese Finches

Male Bengalese finches do not normally change their vocal patterns in adulthood; song is stereotyped and stable over time. Adult song maintenance requires auditory feedback. If adults are deafened, song will degrade within 1 week. We tested whether feedback of all sound frequencies is required for song maintenance. The avian basilar papilla is tonotopically organized; hair cells in the basal region encode high frequencies, and low frequencies are encoded in progressively apical regions. We restricted the spectral range of feedback available to a bird by killing either auditory hair cells encoding higher frequencies or those encoding both high and low frequencies and documented resultant changes in song. Birds were treated with either Amikacin alone to kill high-frequency hair cells or Amikacin and sound exposure to target hair cells across the entire papilla. During treatment, song was recorded from all birds weekly. After treatment and song recording, evoked-potential audiograms were evaluated on each bird, and papillas were evaluated by scanning electron microscopy. Results showed that hair cell damage over 46-63% of the basal papilla and the corresponding high-frequency hearing loss had no effect on song structure. In birds with hair cell damage extending further into the apical region of the papilla and corresponding low-frequency and high-frequency hearing loss, song degradation occurred within 1 week of beginning treatment and was comparable with degradation after surgical deafening. We conclude that either low-frequency spectral cues or temporal cues via feedback of the song amplitude envelope are sufficient for song maintenance in adult Bengalese finches.

A theoretical study of calcium microdomains in turtle hair cells

Confocal imaging has revealed microdomains of intracellular free Ca2+ in turtle hair cells evoked by depolarizing pulses and has delineated factors affecting the growth and dissipation of such domains. However, imaging experiments have limited spatial and temporal resolution. To extend the range of the results we have developed a three-dimensional model of Ca2+ diffusion in a cylindrical hair cell, allowing part of the Ca2+ influx to occur over a small circular region (radius 0.125-1.0 micron) representing a high-density array of voltage-dependent channels. The model incorporated experimental information about the number of channels, the fixed and mobile Ca2+ buffers, and the Ca2+ extrusion mechanism. A feature of the calculations was the use of a variable grid size depending on the proximity to the Ca2+ channel cluster. The results agreed qualitatively with experimental data on the localization of the Ca2+ transients, although the experimental responses were smaller and slower, which is most likely due to temporal and spatial averaging in the imaging. The model made predictions about 1) the optimal Ca2+ channel number and density within a cluster, 2) the conditions to ensure independence of neighboring clusters, and 3) the influence of the Ca2+ buffers on the kinetics and localization of the microdomains. We suggest that an increase in the mobile Ca2+ buffer concentration in high-frequency hair cells (which possess a larger number of release sites) would allow lower amplitude and faster Ca2+ responses and promote functional independence of the sites.

Response characteristics of mammalian cochlear hair cells

Intracellular recordings were made from the low frequency region (third turn) of the guinea pig cochlea. Response characteristics are compared to gross potentials obtained from the organ of Corti fluid space. Inner hair cells (IHCs) possess relatively low (median, -32 mV) initial membrane potentials, whereas that of outer hair cells (OHCs) is higher (median, -53.5 mV). In response to tone burst stimuli, both cell types produce a combination of AC and DC responses. The latter are depolarizing for IHCs but may be of either polarity for OHCs. In terms of their AC responses, IHCs are about 12 dB more sensitive than OHCs. At low sound levels these cells are more linear than high frequency hair cells (Russell, I. J., and P. M. Sellick (1978) J. Physiol. (Lond.) 284: 261-290), judging from the relation between AC and DC response components. At high sound levels pronounced response saturation is seen. The overall tuning properties of the two hair cell types are rather similar, even though IHCs exhibit low frequency velocity dependence, whereas OHCs are displacement sensitive and the cell membrane time constant is larger for IHCs. In order to fit IHC experimental data it is necessary to assume the presence of an underdamped complex pole above the best frequency. The electrical behavior of the OHC does not disqualify it as a conveyor of auditory information to the central nervous system, even though its primary function may be that of a mechanical effector (evidence summarized by Dallos, P. (1985) in Contemporary Sensory Neurobiology, Alan R. Liss, Inc., New York, pp. 207-230).