Models for mechanoelectrical transduction by hair cells.
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Biomedical subjects
Publications and source records attributed to A J Hudspeth.
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The response properties of hair cells and nerve fibers in the alligator lizard cochlea are frequency selective and tonotopically organized with longitudinal position in the organ. The lengths of the hair-cell hair bundles also vary monotonically with longitudinal position. In this study, quantitative measurements were made of the motion of individual hair bundles in an excised preparation of the cochlea stimulated at auditory frequencies. The angular displacement of hair bundles is frequency selective and tonotopically organized, demonstrating the existence of a micromechanical tuning mechanism.
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The sensory epithelium from the bullfrog's sacculus was mounted between two chambers and stimulated by moving the otolithic membrane with a piezoelectric stimulator. The evoked response was measured as the transepithelial "microphonic" potential or, when the epithelium was voltage clamped, as the microphonic current. Microphonic responses were similar to those recorded in other preparations: the whole organ produced a "2f" response (i.e., a response of a frequency twice that of the stimulus) which could be changed to a single-polarity response by stimulating cells of a single polarity; the response saturated asymmetrically with displacement, producing a rectification; and the amplitude declined at high and low frequencies. To determine the cellular elements responsible for generation of the microphonic potential, the equivalent circuit of the epithelium was estimated from morphological and electrophysiological data, and responses to step displacement stimuli were recorded. Four elements in particular shape the microphonic potential: the complex impedance of the extracellular current path, the saturating displacement-conductance curve, an adaptation mechanism which shifts that curve, and a voltage-dependent K+ conductance in the basolateral hair cell membrane. A quantitative model incorporating these elements accurately reproduces the observed responses.
The receptor current of hair cells from the bullfrog's sacculus was measured by voltage clamp recording across the isolated sensory epithelium. Several hundred hair cells were stimulated en masse by moving the overlying otolithic membrane with a piezoelectrically activated probe. As measured by optical recording of otolithic membrane motion, the step displacement stimuli reached their final amplitudes of up to 1 micrometer within 100 microseconds. The relationship between displacement and steady-state receptor current is an asymmetric, sigmoidal curve about 0.5 micrometer in extent. The time constant of the approach to steady state depends upon the magnitude of the hair bundle displacement and ranges from 100 to 500 microseconds at 4 degrees C; the time course is faster with larger displacements or at higher temperatures. Both the displacement-response curve and the kinetics of the response are changed by alterations in the Ca2+ concentration at the apical surface of the cells. The characteristics of the response are not consistent with simple models for the transduction process that involve enzymatic regulation of channel proteins or diffusible second messengers. Mechanical stimulation is instead posited to act directly by altering the free energy difference between the open and closed forms of the transduction channel, thereby inducing a redistribution between these states. The dependences of the response kinetics on displacement and on temperature suggest that the thermal interconversion between open and closed transduction channels is limited by an enthalpy of activation of about 12 kcal/mol.
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The transduction process of a vertebrate hair cell commences with the application of mechanical stimuli to the hair bundle, a cluster microvillous stereocilia and single axonemal kinocilium. In an effort to determine where within the hair bundle transduction occurs, I have measured extracellular potentials around the hair bundles of mechanically stimulated hair cells from the bullfrog's sacculus. Stimulus-dependent signals up to 17 microV in peak-to-peak amplitude have been found. These appear to be due to the flow of transduction current on the basis of their amplitude, phase, dependence on stimulus size and orientation, proportionality to membrane potential, and sensitivity to an ototoxic antibiotic. The responses are consistently larger near the top of the hair bundle than at its base, suggesting that the transduction apparatus lies at or near the distal ends of the stereocilia.
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Piezoelectric bimorph elements are versatile and inexpensive electromechanical transducers which may be used to construct fast mechanical stimulators and finely controlled micromanipulators. The mechanical stimulators described provide continuously graded displacements ranging from nanometers to about a millimeter, or forces equivalent to 0-7 g. Appropriately designed units can produce small step displacements complete within 100 microseconds. Micromanipulators are described which generate 3-dimensional motion under remote electrical control and which enable positioning within a few tenths of a micrometer. They are sufficiently stable to hold glass microelectrodes for cell penetration or probes for microdissection. The two significant drawbacks of bimorph elements are mechanical resonance and continued movement following displacement, or 'creep", but methods have been developed to compensate for these. A number of methods are available to measure motion of bimorphs with spatial resolution of 10 nm and temporal resolution of 2 microseconds in favorable situations.
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An in vitro preparation of hair cells from the bullfrog sacculus produces a transepithelial microphonic potential in response to well-defined mechanical stimuli. If corrected for the electrical time constant of the epithelium, the response follows a fast stimulus with a 40-microsecond delay at 22 degree C. The short latency and its modest temperature dependence limit possible models for transduction by hair cells.
The vertebrate hair cell is a sensory receptor that responds to mechanical stimulation of its hair bundle, which usually consists of numerous large microvilli (stereocilia) and a single true cilium (the kinocilium). We have examined the roles of these two components of the hair bundle by recording intracellularly from bullfrog saccular hair cells. Detachment of the kinocilium from the hair bundle and deflection of this cilium produces no receptor potentials. Mechanical stimulation of stereocilia, however, elicits responses of normal amplitude and sensitivity. Scanning electron microscopy confirms the assessments of ciliary position made during physiological recording. Stereocilia mediate the transduction process of the vertebrate hair cell, while the kinocilium may serve primarily as a linkage conveying mechanical displacements to the stereocilia.
The short working distance of compound microscope lenses in many cases prevents microelectrode penetrations of cells perpendicular to the cell surface. Bending electrodes very near their tips removes this constraint. A method is described for bending glass microelectrodes with a hot filament while their tips are immersed in a water drop. Immersion protects the fine electrode tips from the heat and provides control over the angle through which the electrodes are bent.
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Hair cells, the primary receptors of the auditory, vestibular, and lateral-line sensory systems, produce electrical signals in response to mechanical stimulation of their apical hair bundles. We employed an in vitro preparation and intracellular recording to investigate the transduction mechanism of hair cells in the sacculus from the inner ear of the bullfrog (Rana catesbeiana). When stimulated directly by mechanical deflection of their hair bundles, these cells gave graded responses up to 15 mV in amplitude; the peak sensitivity was about 20 mV/micron deflection. The depolarizing component of the receptor potential corresponding to stimuli directed towards the kinocilium. Depolarizing responses were associated with a membrane resistance decrease, and hyperpolarizing responses with a resistance increase. Action potentials, possibly calcium spikes, were occasionally evoked in hair cells by mechanical or electrical stimulation.
1. The median eye of the giant barnacle, B. nubilus, comprises four large photoreceptor neurones which are visible under the dissecting microscope for almost their entire length. We have studied the structure of, and the responses to light recorded in, the somata, axons, and terminal regions of these neurones.2. The photoreceptor somata, each 40-70 mum in diameter, extend numerous light-sensitive dendritic processes whose membranes form rhabdomeric microvilli. Recordings from the soma show that dim light evokes a steady, noisy depolarization; brighter light elicits a transient depolarization which decays to a maintained plateau, followed by a hyperpolarization when the light is turned off.3. Light-induced voltage changes spread decrementally along the photoreceptor axons, which average 10 mm in length and 25 mum in diameter. In distal parts of the axon, near the presynaptic terminals, depolarizations and hyperpolarizations can be as large as 50% or more of their values in the soma.4. There is no demonstrable electrical coupling between photoreceptor neurones as shown by simultaneous recordings from two receptor somata or axons.5. Each photoreceptor axon enters the mid line commissure of the supraoesophageal ganglion, bifurcates, and arborizes in a restricted zone of neuropil in each hemiganglion. The large size of the terminal processes of these neurones and their characteristic cytoplasmic inclusions enable one to trace them with the electron microscope as they branch in the neuropil.6. The terminal processes subdivide and end in 1-3 mum diameter branches which are the sites of apparently chemical synapses. Vesicle-containing, presynaptic loci on these processes of the receptor cell are invariably apposed to two post-synaptic processes from cells as yet unidentified.
1. The light-induced electrical responses of barnacle photoreceptors spread decrementally along the cells' axons. The decay of the depolarizing and hyperpolarizing components of the visual signal was studied by recording intracellularly from single receptor axons of the median ocellus of the giant barnacle.2. The resistance of the photoreceptor neurone decreases markedly when the cell is depolarized with respect to its dark resting potential of -60 mV. This rectification results in differential attenuation of the depolarizing and hyperpolarizing components of the visual signal as they spread down the axon. Consequently, the visual signal entering the synaptic region is conspicuously distorted.3. Bathing the photoreceptor axons in sodium-free or calcium-free saline or in isotonic sucrose does not significantly affect the spread of the visual signal to the terminals. Thus the signal is not amplified by an ionic mechanism along the axon.4. Membrane characteristics of the photoreceptor for hyperpolarizing voltage changes were estimated from (a) the ratio of the amplitudes of the visual signals recorded simultaneously in the axon and in the soma, (b) the time constant, and (c) the input resistance of the cell. All three independent measurements are consistent with a length constant 1 to 2 times the total length of the cell (lambda = 10-18 mm) and an unusually high membrane resistivity of about 300 kOmega cm(2). This resistivity enables the receptor potential to spread passively to the terminal region.5. Electron microscopic examination of receptor axons reveals an investment of glial lamellae, but demonstrates neither unusual structures which would lead to a high apparent membrane resistivity, nor junctions between cells which would seal off the extracellular space. Thus the observed high resistivity appears to be an intrinsic property of the receptor membrane.