William E. Brownell

Fine structure of the intracochlear potential field i. the silent current

Field potentials were recorded along radial tracks in scala tympani and scala vestibuli of the guinea-pig cochlea. A current density analysis revealed standing current density profiles that were qualitatively similar between animals and between the second and third cochlear turns. Radial standing current densities were greatest at or near the spiral ligament. All the scala vestibuli current density profiles were scaled versions of one another while the scala tympani current density profiles showed more variability. Acoustic stimuli modulated the standing current and there was a cochlear microphonic current density peak in scala tympani near the organ of Corti. The results are summarized with a current-density field line model, the key element of which is a constant current pumped into scale media by the stria vascularis. The standing potential gradients drive current from each perilymphatic chamber into the spiral ligament en route to the lateral surface of the stria vascularis. The strial current is divided between the receptor cell pathway and leakage pathways. The standing current through the leakage pathways is indirectly modulated by acoustic stimulation through the modulation of the endocochlear potential. The reciprocal modulation of current between hair cell and leakage pathways suggests that the stria vascularis maintains a constant current during acoustic stimulation. The cochlear standing current is similar to the retinal dark current in its importance for sensory transduction but the fact that the silent current is generated by the stria vascularis and not the receptor cells provides significant benefits for the detection of mechanical stimuli.

Micropipette aspiration on the outer hair cell lateral wall.

The mechanical properties of the lateral wall of the guinea pig cochlear outer hair cell were studied using the micropipette aspiration technique. A fire-polished micropipette with an inner diameter of approximately 4 microm was brought into contact with the lateral wall and negative pressure was applied. The resulting deformation of the lateral wall was recorded on videotape and subjected to morphometric analysis. The relation between the length of the aspirated portion of the cell and aspiration pressure is characterized by the stiffness parameter, K(s) = 1.07 +/- 0.24 (SD) dyn/cm (n = 14). Values of K(s) do not correlate with the original cell length, which ranges from 29 to 74 microm. Theoretical analysis based on elastic shell theory applied to the experimental data yields an estimate of the effective elastic shear modulus, mu = 15.4 +/- 3.3 dyn/cm. These data were obtained at subcritical aspiration pressures, typically less than 10 cm H2O. After reaching a critical (vesiculation) pressure, the cytoplasmic membrane appeared to separate from the underlying structures, a vesicle with a length of 10-20 microm was formed, and the cytoplasmic membrane resealed. This vesiculation process was repeated until a cell-specific limit was reached and no more vesicles were formed. Over 20 vesicles were formed from the longest cells in the experiment.

Characterization of the outer hair cell's lateral wall membranes

We examined the properties of outer hair cell (OHC) lateral wall membranes by application of 2 fluorescent membrane probes. The markers, C6-NBD-Ceramide and DiOC6, have been used in other cell types to label Golgi apparatus and endoplasmic reticulum, respectively. In living isolated OHCs NBD-Ceramide demonstrated uninterrupted fluorescence along the OHC lateral wall, while DiOC6 labeling proved punctate and notably less uniform in this region. In aldehyde-fixed isolated OHCs both probes exhibited distinct, continuous lateral wall fluorescence. Fixed preparations of the organ of Corti labeled with each probe demonstrated diffuse fluorescence throughout the inner hair cell cytoplasm unlike the uniform, circumferential lateral wall fluorescence seen in OHCs. OHCs exposed to salicylate following NBD-Ceramide labeling displayed patchy, less distinct labeling along the OHC lateral wall. The thickness of lateral wall fluorescence in salicylate exposed cells was 49% greater than control OHCs. We interpreted the salicylate induced change in lateral wall labeling as a fluorescent representation of previously described ultrastructural dilatation and vesiculation of the subsurface cisternae. The distribution of these 2 fluorescent probes along OHC lateral wall membranes suggests that the OHCs subsurface cisternae are neither Golgi nor ER, but share characteristics of both.

Outer Hair Cell Motility and Cochlear Frequency Selectivity

There is mounting evidence that the physiologically vulnerable sensitivity and frequency selectivity of cochlear partition movement (Khanna and Leonard, 1982; Sellick et al., 1982) results from outer hair cell (OHC) bidirectional transduction. These sensory receptors appear not only capable of converting acoustic energy into neural energy (mechano- electrical transduction) but possess effector abilities as well (electromechanical transduction). The first experimental evidence for cochlear bidirectional transduction came from Kemp’s (1978) observation that acoustic energy of cochlear origin can be measured in the external ear canal. Crossed olivo-cochlear bundle (COCB) stimulation has been shown to modulate the magnitude of Kemp’s ota-acoustic emissions (Mountain, 1980; Siegel and Kim, 1982) and to change inner hair cell receptor potentials but not their membrane impedance (Brown and Nutall, 1984). Both types of COCB experiment provide indirect evidence for OHC involvement in the generation of mechanical energy. Recent demonstrations of a motile response of OHC to electrical (Brownell, 1984; Brownell et al., 1985, 1986; Ashmore and Brownell, 1986; Kachar et al, 1986; Evans et al., 1986) and chemical stimuli (Goldstein and Mizukoshi, 1967; Brownell, 1984; Brownell et al., 1985; Flock et al., 1985; Zenner, et al., 1986; Evans et al., 1986) provide direct evidence for electro- and chemo-mechanical transduction by OHCs.

Potential distribution for a spheroidal cell having a conductive membrane in an electric field

When a cell is situated in a uniform electric field, the field is modified due to the relatively low conductance of the cell membrane compared to that of the surrounding fluids. In certain cases, such as in the estimation of internal and external electrokinetic forces, one requires a means of estimating the magnitude of the electric field inside and outside the cell. Most treatments consider the case when the membrane has zero conductivity, or the case of only a spherical cell. The authors solve Laplaces equation for the electric potential distribution inside and outside a cell having a prolate spheroidal shape and having a membrane with a finite, nonzero conductivity.

Measurements and a model of the outer hair cell hydraulic conductivity

The hydraulic conductivity of the cochlear outer hair cell (OHC) is central to the maintenance of the positive intracellular pressure necessary for its function as the cochlear amplifier. A mathematical model of osmotic water transport across the OHC membrane is formulated. The model relates the OHC hydraulic conductivity, Lp, to the rate of volume change in response to osmotic stimuli. Lp is evaluated from osmotic experiments in which isolated OHCs are exposed to an hypotonic solution. The rate of volume increase in response to the hypotonic challenge was determined by a morphometric analysis of video images of cells. Lp was found to be about 10(-14) m s-1 Pa-1 or equivalently, Pf approximately 10(-4) cm s-1. This is on the low side of values reported for different lipid bilayers and is 2 orders of magnitude lower than the hydraulic conductivity of red blood cells. The relation of the low OHC hydraulic conductivity to the composition and morphology of its membranes is discussed.

Outer hair cell length changes in an external electric field. I. The role of intracellular electro‐osmotically generated pressure gradients

Brownell et al. [Science 227, 194-196 (1985)] observed that an isolated, cylindrically shaped cochlear outer hair cell can change its length when an electric field is applied. In their experiments, the cell was fixed at one end, and located between two electrodes which lie on the cell axis but were positioned far from the cell. Kachar et al. [Nature 322, 365-368 (1986)] had suggested that the cells electrically evoked elongation could be caused by pressure gradients resulting from electro-osmosis of the intracellular fluid. A mathematical model is developed which predicts the length change that would result from electro-osmotically generated pressure gradients inside the cell. Estimated parameter values are included to demonstrate that the pressures generated by electro-osmosis inside the cell would result in elongations that are at least two orders of magnitude below the experimentally measured values.

Slow Electrically and Chemically Evoked Volume Changes in Guinea Pig Outer Hair Cells

Our understanding of how the mammalian inner ear converts the mechanical vibrations of sound into neural energy has undergone a fundamental change during the past decade. Nearly ten years of experimental confirmation has convinced the hearing science community that the organ of Corti can produce sound as well as receive it. The most conspicuous evidence for this ability is that sound from the cochlea, or otoacoustic emissions, can be measured in the ear canal. The ear is thought to generate acoustic energy in order to better perform its role of spectrally analyzing the sound it receives (Patuzzi & Robertson, 1988). The inner ear can be considered to be an array of mechanical filters. Each element of this array vibrates at a magnitude determined by the amount of energy the ear receives in the frequency band to which that element is sensitive. A mechanical energy source in each of the elements results in each becoming an active, as opposed to a passive, mechanical filter. An active filter is both more sensitive and faster than a passive filter with the same bandwidth. The processing of auditory information requires speed and mammals appear to have evolved a mechanism that results in narrowly tuned active mechanical filtering. The inherent nonlinearity and instability of this feedback mechanism is thought to be the origin of spontaneous and evoked otoacoustic emissions. The cells responsible for generating the acoustic energy are the outer hair cells. There is one row of around 3000 inner and three rows of around 12000 outer hair cells in organ of Corti. The cylindrical outer hair cells possess a collection of flattened membranous sacs immediately below the cytoplasmic membrane of their lateral wall. These comprise an organelle called the laminated cisternal system that appears unique among eukaryotic cells and may be related to the mechanism that drives the cell’s distinctive electromotile response. Stereocilia-mediated mechanoelectrical modulation of intracochlear currents is thought to elicit electrically evoked outer hair cell length changes at acoustic frequencies and thereby provide for the active filtering that characterizes cochlear transduction.

Outer hair cell length changes in an external electric field. II. The role of electrokinetic forces on the cell surface

An isolated cochlear outer hair cell can elongate or shorten when electrically stimulated, as discovered by Brownell et al. [Science 227, 194-196 (1985)]. In their experiments, the cylindrically shaped cell was fixed at one end, and was positioned between two electrodes which lie on the cell axis, but were far from the cell (transcellular stimulation). A model is developed to predict the component of the cells elongation which arises from only electrokinetic phenomena. Outside the cell, electro-osmosis produces a drag on the lateral wall which almost exactly balances the electrophoretic force. In contrast to previous theories, we find that the electrokinetic response is governed by the free end of the cell, not the lateral wall. If the surface charge density of the free end lies between -0.004 and -0.07 C/m2 (corresponding to the zeta potential between -5 and -60 mV), then our model predicts elongations that are comparable in magnitude to experimentally measured values.

Stimulated volume changes in mammalian outer hair cells

Outer hair cell volume increases in response to superfusion with solutions in which sodium has been replaced with either potassium or sucrose are reported. Volume loss in response to sustained electrical depolarization as well as ototoxic concentrations of salicylate is also described. These stimulated volume changes are attributed to changes in the hydrostatic pressure of the cytoplasm and may involve the mechanisms that regulate outer hair cell turgidity. The loss of turgor and consequent decreases in the fast electromotile response observed with salicylate is consistent with the hearing loss and loss of evoked otoacoustic emissions reported with the ingestion of ototoxic doses of aspirin.<<ETX>>