Jonathan Ashmore

Cochlear Outer Hair Cell Motility

Normal hearing depends on sound amplification within the mammalian cochlea. The amplification, without which the auditory system is effectively deaf, can be traced to the correct functioning of a group of motile sensory hair cells, the outer hair cells of the cochlea. Acting like motor cells, outer hair cells produce forces that are driven by graded changes in membrane potential. The forces depend on the presence of a motor protein in the lateral membrane of the cells. This protein, known as prestin, is a member of a transporter superfamily SLC26. The functional and structural properties of prestin are described in this review. Whether outer hair cell motility might account for sound amplification at all frequencies is also a critical question and is reviewed here.

How well do we understand the cochlea

As sensory cells, hair cells within the mammalian inner ear convert sounds into receptor potentials when their projecting stereocilia are deflected. The organ of Corti of the cochlea contains two types of hair cell, inner and outer hair cells, which differ in function. It has been appreciated for over two decades that although inner hair cells act as the primary receptor cell for the auditory system, the outer hair cells can also act as motor cells. Outer hair cells respond to variation in potential, and change length at rates unequalled by other motile cells. The forces generated by outer hair cells are capable of altering the delicate mechanics of the cochlear partition, increasing hearing sensitivity and frequency selectivity. The discovery of such hair-cell motility has modified the view of the cochlea as a simple frequency analyser into one where it is an active non-linear filter that allows only the prominent features of acoustic signals to be transmitted to the acoustic nerve by the inner hair cells. In this view, such frequency selectivity arises through the suppression of adjacent frequencies, a mechanical effect equivalent to lateral inhibition in neural structures. These processes are explained by the interplay between the hydrodynamic interactions among different parts of the cochlear partition and the effective non-linear behaviour of the cell motor.

The remarkable cochlear amplifier

This composite article is intended to give the experts in the field of cochlear mechanics an opportunity to voice their personal opinion on the one mechanism they believe dominates cochlear amplification in mammals. A collection of these ideas are presented here for the auditory community and others interested in the cochlear amplifier. Each expert has given their own personal view on the topic and at the end of their commentary they have suggested several experiments that would be required for the decisive mechanism underlying the cochlear amplifier. These experiments are presently lacking but if successfully performed would have an enormous impact on our understanding of the cochlear amplifier.

Responses of rod‐bipolar cells in the dark‐adapted retina of the dogfish, Scyliorhinus canicula

1. Responses to light were recorded from bipolar cells in the retina of the dogfish, Scyliorhinus canicula, under dark‐adapted conditions. The identity of the cells was confirmed by Procion Yellow staining.

Fast vesicle replenishment allows indefatigable signalling at the first auditory synapse.

Ribbon-type synapses in inner hair cells of the mammalian cochlea encode the complexity of auditory signals by fast and tonic release through fusion of neurotransmitter-containing vesicles. At any instant, only about 100 vesicles are tethered to the synaptic ribbon, and about 14 of these are docked to the plasma membrane, constituting the readily releasable pool. Although this pool contains about the same number of vesicles as that of conventional synapses, ribbon release sites operate at rates of about two orders of magnitude higher and with submillisecond precision. How these sites replenish their vesicles so efficiently remains unclear. We show here, using two-photon imaging of single release sites in the intact cochlea, that preformed vesicles derived from cytoplasmic vesicle-generating compartments participate in fast release and replenishment. Vesicles were released at a maximal initial rate of 3 per millisecond during a depolarizing pulse, and were replenished at a rate of 1.9 per millisecond. We propose that such rapid resupply of vesicles enables temporally precise and sustained release rates. This may explain how the first auditory synapse can encode with indefatigable precision without having to rely on the slow, local endocytic vesicle cycle.

TRPC3 and TRPC6 are essential for normal mechanotransduction in subsets of sensory neurons and cochlear hair cells

Summary Transient receptor potential (TRP) channels TRPC3 and TRPC6 are expressed in both sensory neurons and cochlear hair cells. Deletion of TRPC3 or TRPC6 in mice caused no behavioural phenotype, although loss of TRPC3 caused a shift of rapidly adapting (RA) mechanosensitive currents to intermediate-adapting currents in dorsal root ganglion sensory neurons. Deletion of both TRPC3 and TRPC6 caused deficits in light touch and silenced half of small-diameter sensory neurons expressing mechanically activated RA currents. Double TRPC3/TRPC6 knock-out mice also showed hearing impairment, vestibular deficits and defective auditory brain stem responses to high-frequency sounds. Basal, but not apical, cochlear outer hair cells lost more than 75 per cent of their responses to mechanical stimulation. FM1-43-sensitive mechanically gated currents were induced when TRPC3 and TRPC6 were co-expressed in sensory neuron cell lines. TRPC3 and TRPC6 are thus required for the normal function of cells involved in touch and hearing, and are potential components of mechanotransducing complexes.

Patch clamped responses from outer hair cells in the intact adult organ of Corti.

Outer hair cells (OHCs) from the mammalian cochlea act as both sensory cells and motor cells. We report here whole-cell tight seal recordings of OHC activity in their natural embedding tissue, the intact organ of Corti, using a temporal bone preparation. The mean cell resting potential, −76±4 mV (n=19) and input conductance (10±3 nS at −70 mV) of third turn hair cells were significantly lower than have been found in isolated cells. Two main K+ currents in the cell were identified. One current, activated positive to −100 mV, was reduced by 5 mM BaCl2. The other current, activated above −40 mV, was reduced by 100 μM 4-aminopyridine (4-AP) and by 30 mM tetraethylammonium (TEA). Both of these currents have been also identified in recordings reported from isolated cells. On stepping to different membrane potentials, cells imaged in the organ of Corti changed length by an amount large enough to cause visible distortions in neighbouring cells. By quantifying such distortions we estimate that the forces generated by OHCs can account for the enhanced response to sound required by the cochlear amplifier.

Purinergic control of intercellular communication between Hensen's cells of the guinea-pig cochlea.

1 Hensens cells in the isolated cochlea were stimulated by extracellular adenosine 5′‐triphosphate (ATP) applied to their endolymphatic surface while changes in membrane current and intracellular calcium concentration ([Ca2+]i) were measured simultaneously. The response consisted of (i) an initial rapid inward current accompanied by elevation of the [Ca2+]i, (ii) a more slowly rising inward current accompanied by a rise of the [Ca2+]i and (iii) a slowly developing reduction of input conductance. 2 The slower responses were maintained in the absence of extracellular Ca2+. Similar responses were produced by increasing the [Ca2+]i via UV flash photolysis of intracellular d‐myo‐inositol 1,4,5‐trisphosphate, P4(5)‐(1‐(2‐nitrophenyl)ethyl) ester (caged InsP3) loaded at pipette concentrations of 8–16 μm. 3 The slow inward current, reversing around 0 mV, was blocked by 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS). 4 Bath application of U‐73122 (1 μm), a phospholipase C inhibitor, eliminated the slow Ca2+‐release component of the response to ATP. It is proposed that the effects of ATP are mediated by the co‐activation of ionotropic P2X and metabotropic P2Y receptors. 5 Immunohistochemistry using light and electron microscopy revealed that inositol 1,4,5‐trisphosphate (InsP3) receptors delineate a network within the cells. 6 The coupling ratio (CR) between cell pairs measured in dual patch‐clamp recordings was 0.356 ± 0.024. The coupling reversibly decreased to 51 % of the control within 2 min of applying 100 μm ATP. Flash photolysis of 32 μm intracellular caged InsP3 and 1 mm caged Ca2+ reduced CR to 42 and 62 % of the control, respectively. 7 We propose that endolymphatic ATP via P2X and P2Y receptors can control intercellular communication amongst Hensens cells by reducing gap junction conductance in a Ca2+‐ and InsP3‐dependent manner.

Calcium signalling mediated by the α9 acetylcholine receptor in a cochlear cell line from the Immortomouse

1 We have investigated the characteristics of the α9 acetylcholine receptor (α9AChR) expressed in hair cell precursors in an immortalized cell line UB/OC‐2 developed from the organ of Corti of the transgenic H‐2Kb‐tsA58 mouse (the Immortomouse) using both calcium imaging and whole‐cell recording. 2 Ratiometric measurements of fura‐2 fluorescence revealed an increase of intracellular calcium concentration in cells when challenged with 10 μM ACh. The calcium increase was seen in 66 % of the cells grown at 39 °C in differentiated conditions. A smaller fraction (34 %) of cells grown at 33 °C in proliferative conditions responded. 3 Caffeine (10 mM) elevated cell calcium. In the absence of caffeine, the majority of imaged cells responded only once to ACh. A small proportion (< 2 % of the total) responded with an increase in intracellular calcium to multiple ACh presentations. Pretreatment with caffeine inhibited all calcium responses to ACh. 4 In whole‐cell tight‐seal recordings 10 μM ACh activated an inward, non‐selective cation current. The reversal potential of the ACh‐activated inward current was dependent on the extracellular calcium concentration with an estimated PCa/PNa of 80 for the α9 receptor at physiological calcium levels. 5 The data indicate that ACh activates a calcium‐permeable channel α9AChR in UB/OC‐2 cells and that the channel has a significantly higher calcium permeability than other AChRs. The results indicate that the α9AChR may be able to elevate intracellular calcium levels in hair cells both directly and via store release.

Sugar transport by mammalian members of the SLC26 superfamily of anion-bicarbonate exchangers

The mammalian cochlea contains a population of outer hair cells (OHCs) whose electromotility depends on an assembly of ‘motor’ molecules in the basolateral membrane of the cell. Named ‘prestin’, the molecule is a member of the SLC26 anion transporter superfamily. We show both directly and indirectly that SLC26A5, rat prestin, takes up hexoses when expressed in several cell lines. Direct measurements of labelled fructose transport into COS‐7 cells expessing prestin are reported here. Indirect measurements, using imaging techniques, show that transfected HEK‐293 or CHO‐K1 cells undergo reversible volume changes when exposed to isosmotic glucose‐fructose exchange. The observations are consistent with the sugar transport. A similar transport was observed using a C‐terminal green fluorescent protein (GFP)‐tagged pendrin (SLC26A4) construct. Cells transfected with GFP alone did not respond to sugars. The data are consistent with fructose being transported by prestin with an apparent Km= 24 mm. From the voltage‐dependent capacitance of transfected cells, we estimate that 250 000 prestin molecules were present and hence that the single transport rate is not more than 3000 fructose molecules s−1. Comparison of the transfected cell swelling rates induced by fructose and by osmotic steps indicates that water was co‐transported with sugar. We suggest that the structure of SLC26 family members allows them to act as neutral substrate transporters and may explain observed properties of cochlear hair cells.