Burt N. Evans

First appearance and development of electromotility in neonatal gerbil outer hair cells

With the purpose of pinpointing the time of onset of electromotility, outer hair cells (OHCs) from apical and basal turns of the cochleae of postnatal gerbils, ranging in age from 6 to 19 days, were isolated and drawn into a glass microchamber. Length changes evoked by transcellular electrical stimulation were detected and measured with a photodiode detector. Motile responses first appeared in 3 out of 14 basal turn OHCs at 7 days after birth (DAB). At 8 DAB, 3 out of 13 apical turn cells also responded to the electrical stimulation. By 12 DAB, all the OHCs from both turns showed motile responses. Input-output functions relating applied stimulus and change in cell length revealed that the motile response threshold improved from 7 DAB to 12 DAB and the response amplitude kept increasing from 7 DAB until 13-14 DAB, when mature amplitudes were reached. Measurements of OHC length revealed only minor changes in basal turn hair cell length while apical hair cells continued to elongate until approximately 16 DAB. Since the onset of auditory function in gerbils occurs around 12 DAB and fine tuning develops between 14 and 17 DAB, our results suggest that the onset of OHC motility occurs earlier than that of auditory function and the maturation of the motility amplitude occurred earlier than the development of fine tuning. The maturation of OHC motility and the development of otoacoustic emissions are also compared and discussed.

Morphology of the unfixed cochlea

Our knowledge of cochlear geometry is based largely upon anatomical observations derived from fixed, dehydrated, embedded and/or sputter-coated material. We have now developed a novel preparation, the hemicochlea, where for the first time living cochlear structures can be observed in situ and from a radial perspective. The experiments were performed on the Mongolian gerbil. Ion substitution experiments suggest that no significant swelling or shrinkage occurs when the preparation is bathed in normal culture medium, so long as calcium concentration is kept at endolymph-like (20 microM) levels. The tectorial membrane-reticular lamina relationship appears to remain well preserved. Hensens stripe maintains a close relationship with the inner hair cell stereociliary bundle, unless the mechanical coupling becomes disturbed. In addition, standard fixation and/or dehydration procedures are used to quantify changes due to shrinkage artifacts. Various morphometric gradients are examined in unfixed specimens from apical, middle, and basal turns.

Outer hair cell electromotility: The sensitivity and vulnerability of the DC component

A technique was devised in order to study the fast electromechanical length changes of outer hair cells at low stimulus levels. Solitary outer hair cells were drawn into a glass microchamber. Length changes were evoked by the application of transcellular potentials and were detected with a photodiode. The method is non-invasive to the cell and offers superior sensitivity and stability for the recording of cell length changes. The function relating command voltage and cell length (V-delta L) change was determined. A nonlinearity, consisting of a DC component superimposed on the AC response, was shown to be present at the lowest stimulus levels measured. The nonlinearity was sensitive to imposed electrical bias as well as vulnerable to overstimulation. The observations are interpreted in reference to the V-delta L function. A parallel is suggested between the nonlinearity seen in the mechanical response and that observed in the responses of the intact cochlea.

Asymmetries in Motile Responses of Outer Hair Cells in Simulated in Vivo Conditions

It is now well known that electrical stimulation of isolated mammalian outer hair cells results in a motile response (Brownell, 1983;Ashmore,1987;Zenneret al.,1987). The fast response component is apparently capable of following a sinusoidal current stimulus in the audio frequency range (Ashmore, 1987; Zenner et al., 1987). Fast motility is a robust response that has been shown under conditions that are distinctly non-physiological. In all experiments reported thus far, all cell surfaces were bathed in a high-sodium medium. Of course, under in vivo conditions only the basolateral cell membrane is exposed to perilymph (high Na+) while the hair-bearing apical end is bathed in endolymph (high K+). We demonstrate here that isolated cells show two kinds of rectification in their motility with sinusoidal current stimuli, that there are quantitative differences in the responses as a function of cell length, and that these phenomena persist when the apical and basolateral cell surfaces are exposed to their proper biochemical environment.

Calibration of photodiode measurements of cell motion by a transmission optical lever method.

A method of calibrating photodiode measurements of cell motion is described. A transparent glass plane is inserted perpendicular to the light path, in front of the photodiode. When the glass plane is rotated, changing the angle between the plane and the light path, the image is displaced. The rotatable glass plane acts as an optical lever to simulate cell motion. The optical lever is easy to construct and offers excellent linearity and accuracy.

The Nonlinearity of Outer Hair Cell Motility: Implications for Cochlear Physiology and Pathology

It is now clear that the motile responses of isolated outer hair cells (OHCs) in vitro are nonlinear (Evans, 1988; Evans et al., 1989, 1990; Santos-Sacchi, 1989). To further our understanding of the nonlinearity and its physiological significance, we have developed a novel preparation (Evans et al., 1989) that offers several improvements over methods previously used in motility studies. Isolated guinea pig OHCs were prepared by enzymatic digestion in papain followed by gentle trituration. The experimental medium, Leibovitz’s L-15 (Gibco), was buffered with 15 mM HEPES, and adjusted to pH 7.35, 320 mOsm. Cells were drawn up by gentle suction into a glass pipette that was heat-polished to a diameter close to that of a hair cell (Fig. 1). The pipette made a resistive seal with the cell body, forming a microchamber. Voltage commands were applied across the cell and the preparation resistance monitored using a current-to-voltage converter. Isolated OHCs were maintained in a separate reservoir and individually transferred to the experimental chamber using a wide-bore suction pipette.

Laser selective microablation of sensitized intracellular components within auditory receptor cells

A laser system can be coupled to a light microscope for laser microbeam ablation and trapping of single cells in vitro. We have extended this technology by sensitization of target structures with vital dyes to provide selective ablation of specific subcellular components. Isolated auditory receptor cells (outer hair cells, OHCs) are known to elongate and contract in response to electrical, chemical and mechanical stimulation. Various intracellular structures are candidate components mediating motility of OHCs, but the exact mechanism(s) is currently unknown. In ongoing studies of OHC motility, we have used the microbeam for selective ablation of lateral wall components and of an axial cytoskeletal core that extends from the nucleus to the cell apex. Both the area beneath the subsurface cistemae of the lateral wall and the core are rich in mitochondria. OHCs isolated from guinea pig cochlea are suspended in L- 15 medium containing 2.0 (mu) M Rhodamine 123, a porphyrin with an affinity for mitochondria. A spark-pumped nitrogen laser pumping a dye cell (Coumarin 500) was aligned on the optical axis of a Nikon Optiphot-2 to produce a 3 ns, 0.5 - 10 micrometers spot (diameter above ablation threshold w/50X water immersion, N.A. 0.8), and energy at the target approximately equals 10 (mu) J/pulse. At short incubation times in Rh123 irradiation caused local blebbing or bulging of cytoplastic membrane and thus loss of the OHCs cylindrical shape. At longer Rh123 incubation times when the central axis of the cell was targeted we observed cytoplasmic clearing, immediate cell elongation (approximately equals 5%) and clumping of core material at nuclear and apical attachments. Experiments are underway to examine the significance of these preliminary observations.

The nature of the generator of outer hair cell motility

Despite the lack of obvious substrates underlying the electrokinetic shape changes of mammalian outer hair cells (OHCs), much can be learned about the mechanism when cells are electrically and mechanically partitioned in a glass microchamber. The results of such studies employing photometric as well as video analysis support the notion that the cellular motor consists of a large number of small, independent elements restricted to the supranuclear region of the cells cortex. The length changes measured in OHCs are highly nonlinear. They are subject to manipulation as well as vulnerable to overstimulation. Thus the mechanical response resembles the mechanism underlying sharp tuning in the intact cochlea. Interestingly, such nonlinearities can be shown to be inherent to each voltage‐sensing element. The whole‐cell movement is thus effectively the cumulative response of many such elements, each driven in parallel by the local transmembrane potential along the length of the cell.