Mapping the distribution of outer hair cell voltage-dependent conductances by electrical amputation

The mammalian outer hair cell (OHC) functions not only as sensory receptor, but also as mechanical effector; this unique union is believed to enhance our ability to discriminate among acoustic frequencies, especially in the kilohertz range. An electrical technique designed to isolate restricted portions of the plasma membrane was used to map the distribution of voltage-dependent conductances along the cylindrical extent of the cell. We show that three voltage-dependent currents, outward K, I(K,n), and I(Ca) are localized to the basal, synaptic pole of the OHC. Previously we showed that the lateral membrane of the OHC harbors a dense population of voltage sensor-motor elements responsible for OHC motility. This segregation of membrane molecules may have important implications for auditory function. The distribution of OHC conductances will influence the cable properties of the cell, thereby potentially controlling the voltage magnitudes experienced by the motility voltage sensors in the lateral membrane, and thus the output of the "cochlear amplifier."     

Frequency tuning of basilar membrane and auditory nerve fibers in the same cochleae

Responses to tones of a basilar membrane site and of auditory nerve fibers innervating neighboring inner hair cells were recorded in the same cochleae in chinchillas. At near-threshold stimulus levels, the frequency tuning of auditory nerve fibers closely paralleled that of basilar membrane displacement modified by high-pass filtering, indicating that only relatively minor signal transformations intervene between mechanical vibration and auditory nerve excitation. This finding establishes that cochlear frequency selectivity in chinchillas (and probably in mammals in general) is fully expressed in the vibrations of the basilar membrane and renders unnecessary additional ("second") filters, such as those present in the hair cells of the cochleae of reptiles.     

A potassium current in guinea-pig outer hair cells activated by ion channel blocker DCDPC

Fenamate compounds have been reported to inhibit ion channels in a number of tissues, including a non-selective cation channel in the mammalian outer hair cell (OHC). We have further investigated the effects of 3'-5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) on OHC currents using the whole-cell configuration of the patch clamp technique. Extracellular application of 10 microM DCDPC rapidly and reversibly activated an inward current at hyperpolarized potentials. The DCDPC-activated current appeared in the shorter OHCs from the basal turns of the cochlea. The reversal potential of the inward current was dependent on the external K+ ion concentration. An outwardly rectifying K+ current, found predominantly in OHCs from apical turns, was reversibly inhibited by DCDPC. These results suggest that DCDPC has a significant effect on OHC physiology at all tonotopic locations along the basilar membrane and so may have implications for cochlear function during fenamate intake.     

A model for the responses of low-frequency auditory-nerve fibers in cat

A computational model was developed for the responses of low-frequency auditory-nerve (AN) fibers in cat. The goal was to produce realistic temporal response properties and average discharge rates in response to simple and complex stimuli. Temporal and average-rate properties of AN responses change as a function of sound-pressure level due to nonlinearities in the auditory periphery. The input stage of the AN model is a narrow-band filter that simulates the mechanical tuning of the basilar membrane. The parameters of this filter vary continuously as a function of stimulus level via a feedback mechanism, simulating the compressive nonlinearity associated with the mechanics of the basilar membrane. A memoryless, saturating nonlinearity and two low-pass filters simulate transduction and membrane properties of the inner hair cell (IHC). A diffusion model for the IHC-AN synapse introduces adaptation. Finally, a nonhomogeneous Poisson process, modified by absolute and relative refractoriness, provides the output discharge times. Responses to several different stimuli are presented. These responses illustrate nonlinear temporal response properties that cannot be achieved with linear models for AN fibers.     

Postnatal development of the organ of Corti in cats: a light microscopic morphometric study

This study quantitatively characterizes the development of the major morphological features of the organ of Corti during the first 2 weeks postnatal, the period when the cat auditory system makes the transition from being essentially non-functional to having nearly adult-like responses. Four groups of kittens (n = 3) were studied at one day postnatal (P1), P5, P10, P15, and compared to adults. Measurements were made of the organ of Corti at 3 cochlear locations: 20%, 60% and 85% of basilar membrane length from the base cochlear locations which in the adult correspond to best frequencies of approximately 20 kHz, 2 kHz and 500 Hz, respectively. In addition, measurements of basilar membrane length and opening of the tunnel of Corti were made in 20 cochlear specimens from kittens aged P0-P6. Results indicate that: (i) at P0 the basilar membrane has attained adult length, and the tunnel of Corti is open over approximately the basal one-half of the cochlea; (ii) the initial opening of the tunnel of Corti occurs at a site about 4 mm from the cochlear base (best frequency of approximately 25 kHz in the adult cochlea); (iii) the thickness of the tympanic cell layer decreases markedly at the basal 20-kHz location; (iv) the areas of the tunnel of Corti and space of Nuel and the angulation of the inner hair cells (IHC) relative to the basilar membrane all show marked postnatal increases at both the middle and apical locations; (v) IHC are nearly adult-like in length and shape at birth, whereas the OHC (at 2-kHz and 500-Hz locations) undergo marked postnatal changes; (vi) disappearance of the marginal pillars and maturation of the supporting cells are not yet complete by P15.     

Distortion-product otoacoustic emissions in kanamycin-treated guinea pig cochlea

Measurement of distortion-product otoacoustic emissions (DPOAE) is widely accepted as one of the most valuable tools for evaluating the frequency of specific cochlear pathology. Previous studies have revealed that distortion-product levels in the ear canal are definitely correlated with degree of damage in the cochlea. However, there seem to be no clear data of help in predicting the distribution and grade of damage in the cochlea quantitatively on the basis of the results of this non-invasive test. The present study is designed to assess correlations between degree of outer hair cell (OHC) damage by a potent ototoxic antibiotic, kanamycin, and DPOAE levels at the characteristic frequency at the site. Guinea pigs were used after daily intramuscular administration of kanamycin for 7 or 10 days. DPOAE levels were measured using a system (CUBDIS: Etymotic Research) with 78 frequency combinations of iso-intensity primaries from 0.5kHz to 16kHz of f2. The frequency ratio (f2/f1) was set at 1.2. Distortion-product level plots versus f2 (DP-grams) were constructed. The integrity of the OHC system was evaluated histologically by the succinic dehydrogenase (SDH) method under a light microscope. Cochleograms were constructed by calculating percentages of intact OHCs along the basilar membrane in 1-mm blocks. The DP-grams and the histopathological cochleograms showed essentially identical patterns in the kanamycin-damaged guinea pig cochlea. The results suggest that: 1) The generation of DPOAE requires functioning OHCs. 2) DPOAE measurement provides information allowing prediction of OHC damage distribution in the cochlea without histological investigations. 3) Careful setting of primary levels and other parameters is necessary to reliably predict the pathology. 4) Attempts to detect of minimal OHC damage could fail. 5) DPOAE seem very useful for monitoring cochlear function in clinically.     

Ultrastructural localisation of spectrin in sensory and supporting cells of guinea-pig organ of Corti

Spectrin is a cytoskeletal protein found in the cortex of many cell types. It is known to occur in cochlear outer hair cells (OHCs) with previous immunoelectron microscopical studies showing that it is located in the cuticular plate and the cortical lattice. The latter is a network of filaments associated with the lateral plasma membrane that is thought to play a role in OHC motility. Spectrin has also been found in inner hair cells (IHCs) and supporting cells using immunofluorescent techniques, but its ultrastructural distribution in these cells has not yet been described. This has, therefore, been investigated using a monoclonal antibody to alpha-spectrin in conjunction with pre- and post-embedding immunogold labelling for transmission electron microscopy. Labelling was found in a meshwork of filaments beneath the plasma membranes of both IHCs and supporting cells and, in pillar cells, close to microtubule/microfilament arrays. It was also found in association with the stereocilia of OHCs and IHCs and, as expected, in the cortical lattice and cuticular plate of OHCs. Thus, spectrin is a general component of cytoskeletal structures involved in maintaining the specialised cell shapes in the organ of Corti and may contribute to the mechanical properties of all the cell types examined.     

The level dependence of response phase: observations from cochlear hair cells

Hair cell responses are recorded from third turn of the guinea pig cochlea in order to define the relationship between hair cell depolarization and position of the basilar membrane. Because the latter is determined locally, using the cochlear microphonic recorded in the organ of Corti (OC) fluid space, no corrections are required to compensate traveling wave and/or synaptic delays. At low levels, inner hair cells (IHC) depolarize near basilar membrane velocity to scala vestibuli reflecting the free standing nature of their stereocilia. At high levels, the time of depolarization changes rapidly from velocity to scala vestibuli to the scala tympani phase of the basilar membrane response. This change in response phase, recorded in the fundamental component of the IHC response, is associated with a decrease in response magnitude. The absence of this behavior in OC and outer hair cell responses implies that basilar membrane mechanics may not be responsible for these response patterns. Because these features are reminiscent of the magnitude notches and the large phase shifts observed in single unit responses at high stimulus levels, they provide the IHC correlates of these phenomena.     

Cochlear outer hair cell bending in an external electric field

We have used a high-resolution motion analysis system to reinvestigate shape changes in isolated guinea pig cochlear outer hair cells (OHCs) evoked by low-frequency (2-3 Hz) external electric stimulation. This phenomenon of electromotility is presumed to result from voltage-dependent structural changes in the lateral plasma membrane of the OHC. In addition to well-known longitudinal movements, OHCs were found to display bending movements when the alternating external electric field gradients were oriented perpendicular to the cylindrical cell body. The peak-to-peak amplitude of the bending movement was found to be as large as 0.7 microm. The specific sulfhydryl reagents, p-chloromercuriphenylsulfonic acid and p-hydroxymercuriphenylsulfonic acid, that suppress electrically evoked longitudinal OHCs movements, also inhibit the bending movements, indicating that these two movements share the same underlying mechanism. The OHC bending is likely to result from an electrical charge separation that produces depolarization of the lateral plasma membrane on one side of the cell and hyperpolarization on the other side. In the cochlea, OHC bending could produce radial distortions in the sensory epithelium and influence the micromechanics of the organ of Corti.     

Neuregulins and erbB receptors at neuromuscular junctions and at agrin-induced postsynaptic-like apparatus in skeletal muscle

This study has characterized the repertoire of the anion exchanger (AE) family members expressed within the guinea pig organ of Corti, the auditory neuroepithelia. Both AE2 and AE3 cDNAs were present, but AE1 cDNA was not detected. The more abundant AE2 was sequenced and its expression characterized in the cochlea. The 3888 base pairs (bp) AE2 sequence, compiled from multiple clones, includes 150 bp of upstream non-coding sequence and 3717 bp of open reading frame encoding a protein of 1238 amino acids. Immunoblot of cochlear homogenate revealed a single AE2-immunoreactive band of Mr 180 kDa. In situ hybridization and immunohistochemical analysis localized AE2 expression to several tissues and cell types within the guinea pig inner ear, including superior half of the spiral ligament and within the interdental cells lining the spiral limbus. However, AE2 was not clearly detected in the outer hair cells (OHC) of the organ of Corti by either immunohistochemistry or in situ hybridization. The results of these studies imply a physiologic role of AE2 in the cochlear homeostasis, but do not support its role as a potential 'motor protein' in mediating the in vitro-observed voltage-gated, ATP-independent OHC motility.     

Acute hyperpolarization and elongation of cochlear outer hair cells on superfusion with cis-platinum

The acute effects of cis-platinum on isolated cochlear outer hair cells (OHC) were investigated with whole-cell patch-clamps and measurements of cell length changes. Our findings demonstrated that cis-platinum reversibly induced a hyperpolarization and cellular elongation. These results suggest that the effects produced are the result of an interaction between cis-platinum and transduction channels in OHC. These acute effects are distinctly different from the chronic, irreversible ones that are followed by death of the OHC. The exact mechanism of these chronic effects remains unknown as yet.     

Hair cell loss from acoustic trauma in chloroquine-treated red, black and albino guinea pigs

In order to further elucidate the relationship between noise-induced hearing loss and pigmentation, a two-factor study was designed. Albino, red and black guinea pigs were divided into controls and chloroquine-treated groups and exposed to 1 kHz noise, 105 dB SPL, for 72 hours. One month later the animals were sacrificed and the loss of hair cells evaluated. The red guinea pigs developed a greater hair cell loss (OHC) in all three OHC rows than black or albino animals. Black and albino groups showed equal amounts of OHC loss. A high dosage of chloroquine seemed to reduce the OHC loss in albino, but not in black or red guinea pigs. The greater OHC loss in red compared with black animals is in accordance with the original hypothesis that melanin protects the inner ear against noise trauma. However, as red guinea pigs developed greater OHC loss than albinos, it is obvious that the original hypothesis needs to be modified to consider also the different melanin types, i.e., the black eumelanin and the red pheomelanin. The present results are interpreted as a toxic interaction in the strial melanocytes between pheomelanin and noise. It is suggested that the pathophysiology of noise-induced hearing loss involves cochlear mechanisms related to radical oxygen species (ROS) as melanin both generates and neutralizes ROS. A hypothesis about a linkage between dopamine, noise trauma and the cochlear melanocyte system is discussed.     

Can the neurotrophic hypothesis explain degeneration and loss of plasticity in mature and ageing autonomic nerves?

The development of Ca-ATPase immunoreactivity in gerbil outer hair cells (OHCs), assayed by immunofluorescence and postembedding immunocytochemistry, is reported here. In the adult, a linear array of label is seen inside the lateral plasma membrane. The ultrastructural distribution of Ca-ATPase near the OHC lateral plasma membrane was examined using immunogold cytochemistry and showed this calcium pumping enzyme to be present throughout the subsurface cisternal complex (SSC), especially near the innermost layers. During development, Ca-ATPase immunoreactivity appeared in patches near the lateral plasma membrane of some OHCs of the third row by 12 days after birth (DAB). By 15-16 DAB, punctate immunoreactivity was detected in the second and first rows. At 20 DAB, immunostaining near OHC lateral plasma membrane was increased, but was less continuous than OHC staining in the adult cochlea. The appearance of Ca-ATPase in OHCs coincides with the onset of auditory function and isolated OHC motility in the gerbil. The ultrastructural demonstration of abundant sites of calcium pumps in the SSC supports a role for this structure in the intracellular storage of calcium. These findings suggest a possible role of Ca-ATPase and the SSC in the regulation of slow motility of OHCs which has been reported to depend on intracellular calcium concentration.     

Pathophysiological mechanisms of hearing loss

An understanding of auditory transduction in the ear can contribute to a better comprehension of the pathophysiological mechanisms which give rise to hearing loss. The incoming sound sets up a mechanical traveling wave which begins at the base and progresses along the basilar membrane, reaching a point of maximal displacement. The region of maximal displacement is a function of stimulus frequency. The mechanical displacement, by directly opening ion channels in the stereocilia of the hair cells, induces changes in the electrical potential of the hair cells. This initial stage is called mechano-electrical transduction, and in the normal ear, is followed by a stage of electro-mechanical transduction based on the ability of the outer hair cells to respond to the electrical changes induced in them with a change in their length. This "electromotility" presumably provides mechanical feedback to the basilar membrane, augmenting its mechanical displacement. This is called the cochlear amplifier, providing the ear with improved sensitivity and frequency discrimination. Most forms of sensori-neural hearing losses (affecting the inner ear) are due to a lesion to some part of this cochlear amplifier (e.g. noise induced hearing loss, ototoxic drugs) and are therefore characterized by auditory threshold elevations and poorer frequency discrimination.     

Postnatal maturation of the organ of Corti in gerbils: morphology and physiological responses

The organ of Corti, the sensory epithelium of hearing in mammals, matures postnatally in the gerbil. Quantitative analyses of the postnatal development of the organ of Corti, including supporting cells and the basilar membrane, were carried out. The morphological study confirmed that maturation of the sensory cells proceeds with a base-to-apex gradient, with the outer hair cells appearing to mature before the inner hair cells. Maturation of the supporting cells and the basilar membrane commenced first in the middle turn. Expansion of the second row of Deiters' cells began at 6 days after birth in the middle turn, before enlargement of the pillar cell heads at 8 days postnatally. Pillar cell head enlargement continued until 20 days postnatally in the middle turn. The tunnel of Corti and spaces of Nuel appeared first in the middle turn between 8 and 10 days postnatally. The maturation of the basilar membrane involved the thickening of the central hyaline layer and a reduction in the epithelial cells on the tympanic aspect. This process continued until about 20 days after birth. The cochlear microphonic potential, whole nerve action potential, and stimulus frequency otoacoustic emissions were recorded from 12 days after birth onward and related to changes in organ of Corti morphology. The results show that changes in the accessory structures continue throughout the period of onset and development of cochlear responses between 12 and 20 days after birth, and may therefore influence the micromechanical responses of the organ of Corti to acoustic stimuli during this period.     

Pathologic mechanoelectric transduction of outer hair cells as the cause of recruitment

It is believed that the sound-induced travelling wave in the mammalian cochlea is enhanced and sharpened by a positive feedback mechanism. This causes the passive linear basilar membrane growth function to become non-linear. The present paper shows that nonlinear basilar membrane vibration is due to the nonlinear growth function of the receptor potential of outer hair cells, which can be described by a 2nd-order Boltzmann function. Since intensity coding in the inner ear depends on an interaction of nonlinear basilar membrane motion and nerve fibers with three different types of synaptic threshold and growth function, the process is directly dependent on an intact mechanoelectrical transduction of outer hair cells. According to the proposed model, a loss in efficiency of outer hair cell mechanoelectrical transduction must lead to both a reduction in gain (i.e., hearing loss) and a linearizing of the response. As a result, once above threshold, the changes of stereociliary displacement, basilar membrane displacement and neural firing rate per unit change of sound intensity must be larger than for the healthy cochlea with its compressive nonlinearity.     

Compression, gain, and nonlinear distortion in an active cochlear model with subpartitions

The propagation of inhomogeneous, weakly nonlinear waves is considered in a cochlear model having two degrees of freedom that represent the transverse motions of the tectorial and basilar membranes within the organ of Corti. It is assumed that nonlinearity arises from the saturation of outer hair cell active force generation. I use multiple scale asymptotics and treat nonlinearity as a correction to a linear hydroelastic wave. The resulting theory is used to explain experimentally observed features of the response of the cochlear partition to a pure tone, including: the amplification of the response in a healthy cochlea vs a dead one; the less than linear growth rate of the response to increasing sound pressure level; and the amount of distortion to be expected at high and low frequencies at basal and apical locations, respectively. I also show that the outer hair cell nonlinearity generates retrograde waves.     

The mechanism and site of action of lidocaine hydrochloride in guinea pig inner ear

Lidocaine was applied to the round window (RW) in order to localize its site of action in the cochlea. Cochlear microphonic (CM), summating potential (SP), and compound action potential (CAP) input/output functions were measured to a 16 kHz tone burst to assess the functional changes of the cochlea. In separate experiments, the effect of lidocaine on the whole cell current of isolated outer hair cells (OHC) was studied. A dose of 2 microliters of 40 mM lidocaine in saline solution, when applied to the RW, caused a small change in all measured variables, indicating a passage of the drug through the RW membrane to sites of action. However, 160 mM of lidocaine further decreased CM, SP, and CAP by a total of 40% from the control. A partial recovery occurred for CM during the 30 min follow-up period. CAP and SP continued to decline. In isolated OHCs, lidocaine decreased the whole cell current in a dose-dependent fashion. The KD for lidocaine effect on OHCs was 7 mM. Our in vivo results indicate that lidocaine affects OHCs and reduces CM, causing a subsequent reduction in SP and CAP. The increased effect of lidocaine on CAP and SP, while CM is recovering, suggests an additional specific effect of lidocaine on the cochlear nerve and/or on inner hair cells. Considering that lidocaine alters OHC current (in isolated hair cells) and that lidocaine does not affect endocochlear potential [Laurikainen et al. Acta Otolaryngol (Stockh) 1991: 112: 800-9], the observed CM changes are most likely due to an in vivo effect on OHCs. Thus, the early effect of lidocaine on the cochlea appears to be due to a significant change in organ of Corti function, rather than to direct anesthesia of the cochlear nerve. Later, an independent effect of the drug may occur on neural tissues in the inner ear.     

Long-term effects of sectioning the olivocochlear bundle in neonatal cats

The olivocochlear bundle (OCB) was cut in neonatal cats to evaluate its role in the development of normal cochlear function. Approximately 1 year after deefferentation, acute auditory nerve fiber (ANF) recordings were made from lesioned animals, lesion shams, and normal controls. The degree of deefferentation was quantified via light microscopic evaluation of the density of OCB fascicles in the tunnel of Corti, and selected cases were analyzed via electron microscopy. In the most successful cases, the deefferentation was virtually complete. ANFs from successfully lesioned animals exhibited significant pathophysiology compared with normals and with other animals in which the surgery failed to interrupt the OCB. Thresholds at the characteristic frequency (CF), the frequency at which ANFs are most sensitive, were elevated across the CF range, with maximal effects for CFs in the 10 kHz region. Frequency threshold or tuning curves displayed reduction of tip-to-tail ratios (the difference between CF and low-frequency "tail" thresholds) and decreased sharpness of tuning. These pathological changes are generally associated with outer hair cell (OHC) damage. However, light microscopic histological analysis showed minimal hair cell loss and no significant differences between normal and deefferented groups. Spontaneous discharge rates (SRs) were lower than normal; however, those fibers with the highest SRs remained more sensitive than those with lower SRs. Findings suggest that the interaction between OC efferents and OHCs early in development may be critical for full expression of active mechanical processes.