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J F Ashmore

Publications and source records attributed to J F Ashmore.

At least 55 records · Page 3Linked to original sources
PubMed1990

Control of intracellular calcium by ATP in isolated outer hair cells of the guinea-pig cochlea.

1. Intracellular calcium levels were monitored in isolated outer hair cells of the guinea-pig cochlea using the calcium-sensitive dye Fura-2. 2. The calcium in the cells was studied during application of ATP externally applied from a pipette. ATP induced a rise of intracellular calcium which could be separated into two components: a rapid rise, peaking in 20 s, localized around the apical end of the cell, and a slower rise, peaking in 50-150 s but spread throughout the cell. The effects were observed with 5, 25 and 100 microM-ATP concentrations. 3. In the absence of external Ca2+, ATP was still able to trigger a rise in Ca2+, but with a longer delay. Under these conditions, the cells did not show the initial rapid Ca2+ rise. The result suggests that ATP can mobilize intracellular stores. 4. A rise in intracellular Ca2+ was also observed when 5 mM-caffeine was applied to the bath. 5. Simultaneous measurements were made of whole-cell currents and intracellular calcium. ATP activated an inward current at resting potentials of -60 mV. Internal Ca2+ levels increased during the inward current. In current-clamped cells Ca2+ levels also increased during the associated depolarization produced by ATP. 6. Adenosine (150 microM) did not produce any measurable inward current. Acetylcholine (ACh, 100 microM-1 mM) produced only a small rise in Ca2+. However, applied simultaneously with ATP, ACh suppressed the rise in intracellular Ca2+ produced by ATP, with the kinetics of a competitive antagonist. 7. Intracellular Ca2+ increased with step depolarizations of the cell above -20 mV during whole-cell clamp. Large rises in Ca2+ were also observed on depolarizing the cell with isotonic KCl. 8. Calcium levels in supporting cells of the organ of Corti were sensitive to ATP. In these cells, rises in intracellular Ca2+ did not require the presence of extracellular Ca2+. 9. It is concluded that the organ of Corti contains receptors for ATP on a variety of the cells. ATP controls a direct entry of Ca2+ through the membrane and also may mobilize intracellular stores.

Acetylcholine↗
PubMed1990

Spectrin, actin and the structure of the cortical lattice in mammalian cochlear outer hair cells.

Mammalian cochlear outer hair cells generate high-frequency forces in response to electrical stimulation. Force generation occurs in the lateral cortex of the cell, which includes the plasma membrane, a two-dimensional 'cortical lattice' of filamentous protein, and a multi-layered membrane system, the lateral cisternae. The cortical lattice is composed of relatively long filaments, 6.7 nm in diameter, which are wound circumferentially about the cell. These filaments are spaced about 42 nm apart and are cross-linked by a second type of filament 3.2 nm in diameter approximately aligned with the longitudinal axis of the cell. The cortical lattice is the only cortical structure that remains after the cell is fully extracted in the detergent Triton X-100 and high-salt solution. It retains the original cylindrical shape of the cell and is reversibly deformable. Antibodies raised against chicken gizzard actin, human blood spectrin and pig brain spectrin all react positively with the extracted lattice viewed using immunofluorescence. Three protein subunits identified in the organ of Corti have approximate molecular weights of 220, 235 and 240K (K = 10(3) Mr) and react with the spectrin antibodies. A structural model of the lattice is proposed in which the circumferential filaments are composed of actin and the cross-linked of spectrin. The model can account for the unusual cylindrical shape of outer hair cells and suggests a mechanism of force generation based upon the elastic and electrostatic properties of spectrin.

Actins↗
PubMed1988

A cytoskeletal spring in cochlear outer hair cells.

Normal hearing in mammals depends on an active mechanical filter, within the cochlea, which separates different sound frequencies before neural encoding. Experiments on the intact cochlea indicate that the critical cellular components underlying the process are probably the outer hair cells which are strategically placed to influence movement of the basilar membrane. This idea is attractive because isolated cells can generate axial forces at acoustic frequencies when electrically stimulated. The mechanical properties of cells are largely determined by structures closely associated with the plasma membrane. We show here, using light and electron microscopy, that beneath this membrane lies a lattice of crosslinked circumferential filaments which are pitched at a mean angle of 15 degrees to the transverse axis of the cell. The lattice is sufficient to retain the shape of the cell following demembranation and mechanical deformation. The structure of the lattice allows it to be described as a coiled helical spring but with longitudinal stiffness primarily determined by the crosslinks. Direct measurements of longitudinal stiffness reported here indicate that the lattice contributes 5-10% of the stiffness. We propose that the 'circumferential lattice' ensures that outer hair cells can act as directed force generators within the organ of Corti, a prerequisite in current descriptions of cochlear micromechanics.

Hair Cells, Auditory↗
PubMed1988

On the mechanism of a high-frequency force generator in outer hair cells isolated from the guinea pig cochlea.

Isolated mammalian outer hair cells elongate or shorten respectively by several micrometres when electrically hyperpolarized or depolarized. The experiments in this paper were designed to locate the force-generating mechanism that drives length changes in outer hair cells, and to determine some of its basic properties. The whole-cell mode of the patch-clamp technique was used to stimulate cells electrically and to perfuse them with specific drugs. The pattern of displacement of cellular organelles, and the relative displacements of the cell base and apex during electrical stimulation with the cell mechanically anchored at various points along its length, suggest that the force-generating mechanism is distributed throughout the length of the cell. Further experiments altering the shape, volume and intracellular pressure of outer hair cells suggest that the mechanism is closely associated with the plasma membrane. These experiments also demonstrate that the characteristic tubular shape of outer hair cells is maintained by membrane-associated structures with elastic properties that enable the cell to return to its original shape after deformation. The mechanism controlling length changes may, therefore, be composed of two elements in parallel, namely a force generating element and a passive elastic element. Inhibitors of ATP synthesis, or the presence of the non-hydrolysable ATP analogue AMP.PNP, perfused into outer hair cells, failed to inhibit length changes. Drugs against actin, including phalloidin, cytochalasin B and cytochalasin D, and against tubulin, including colchicine, nocodazole and colcemid, also failed to inhibit length changes. We conclude that the force-generating mechanism is, therefore, unlike most other forms of cell motility, and possible alternative hypotheses are briefly discussed.

Actins↗
PubMed1988

Temperature-dependence of a fast motile response in isolated outer hair cells of the guinea-pig cochlea.

Isolated outer hair cells (OHC) of the cochlea exhibit a motility which can be driven at acoustic frequencies (Ashmore, 1987). The temperature-dependence of the movement is reported here for the range 20-37 degrees C. OHC are shown to be motile at 37 degrees C. The temperature-dependence is quite weak, with Q10 = 1.33. The observation is difficult to reconcile with mechanisms involving an intermediate biochemical messenger, including those based on actomyosin.

Animals↗
PubMed1988

Hair cells.

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Animals↗
PubMed1987

An electrical resonance in hair cells of the amphibian papilla of the frog Rana temporaria.

Intracellular recordings were made from hair cells in the rostral- and mid-regions of an in vitro preparation of the amphibian papilla from the frog Rana temporaria. In response to small current injections the membrane potential oscillated indicating the presence of an electrical resonator within these hair cells. The properties of this resonance were similar to those of electrical resonance in hair cells of other lower vertebrates (Crawford and Fettiplace, 1981). The frequency of these oscillations depended on the position of the cell within the papilla; the oscillation frequency at the offset of the current pulse appeared to be tonotopically organized. The role of this resonance in relation to the known frequency selectivity and tonotopic organization of this auditory organ is discussed.

Animals↗
PubMed1987

A fast motile response in guinea-pig outer hair cells: the cellular basis of the cochlear amplifier.

1. Outer hair cells from the cochlea of the guinea-pig were isolated and their motile properties studied in short-term culture by the whole-cell variant of the patch recording technique. 2. Cells elongated and shortened when subjected to voltage steps. Cells from both high- and low-frequency regions of the cochlea responded with an elongation when hyperpolarized and a shortening when depolarized. The longitudinal motion of the cell was measured by a differential photosensor capable of responding to motion frequencies 0-40 kHz. 3. Under voltage clamp the length change of the cell was graded with command voltage over a range +/- 2 microns (approximately 4% of the length) for cells from the apical turns of the cochlea. The mean sensitivity of the movement was 2.11 nm/pA injected current, or 19.8 nm/mV membrane polarization. 4. The kinetics of the cell length change during a voltage step were measured. Stimulated at their basal end, cells from the apical (low-frequency) cochlear turns responded with a latency of between 120 and 255 microseconds. The cells thereafter elongated exponentially by a process which could be characterized by three time constants, one with value 240 microseconds, and a second in the range 1.3-2.8 ms. A third time constant with a value 20-40 ms characterized a slower component which may represent osmotic changes. 5. Consistent with the linearity shown to voltage steps, sinusoidal stimulation of the cell generated movements which could be measured at frequencies above 1 kHz. The phase of the movement relative to the stimulus continued to grow with frequency, suggesting the presence of an absolute delay in the response of about 200 microseconds. 6. The electrically stimulated movements were insensitive to the ionic composition of the cell, manipulated by dialysis from the patch pipette. The responses occurred when the major cation was K+ or Na+ in the pipette. Loading the cell with ATP-free solutions or calcium buffers did not inhibit the response. 7. It is concluded that interaction between actin and myosin, although present in the cell, is unlikely to account for the cell motility. Instead, it is proposed that outer hair cell motility is associated with structures in the cell cortex. The implications for cochlear mechanics of such force generation in outer hair cells are discussed.

Animals↗
PubMed1986

Stiffness of sensory hair bundles in the sacculus of the frog.

The stiffness of individual hair bundles of hair cells from the frog sacculus was measured using calibrated quartz probes. For displacements of up to 1 micron in either direction (angular deflections up to +/- 0.13 rad) the stiffness was constant. The stiffness did not depend on whether the bundle was in compression or tension. At first approximation, the stiffness was inversely proportional to the square of the height of application of the force above the apical surface of the hair cell. This is consistent with pivoting of the stereocilia within the hair bundle about their points of insertion into the cuticular plate. The pivotal stiffness of the bundle was approximately proportional to the bundle's cross-sectional area and hence to the number of stereocilia of which it is composed. It is inferred that the contribution of the kinocilium to the total bundle stiffness is small. It is concluded that applied forces are shared almost equally amongst all stereocilia, that there is relative shear between neighbouring stereocilia during bundle deflection and that each stereocilium contributes a pivotal stiffness of 0.49 +/- 0.15 X 10(-15) N X m X rad-1. The measured stiffness of the stereocilium is consistent with a structure which bends mainly at the tapering insertion point. The data are also consistent with little cross-linking here between actin filaments. The nature of the links between stereocilia in the hair bundle is also discussed.

Animals↗
PubMed1983

Kinetics of synaptic transmission from photoreceptors to horizontal and bipolar cells in turtle retina.

The kinetics of synaptic transfer from photoreceptors to second order neurons have been determined in the retina of the snapping turtle. Analysis of the fluctuations resulting from the random release of cone transmitter molecules reveals that the action on postsynaptic channels of the depolarizing bipolar cells is prolonged by approximately 10 times compared to the action on the hyperpolarizing bipolar cells. Determination of the cone and rod to horizontal cell impulse response shows that the kinetics of the cone/H1AT synapse is approximately 8-10 times shorter than the rod/H1AT synapse. These results indicate that the cone driven, sign preserving synapses exhibit comparable temporal characteristics.

Animals↗
PubMed1983

An analysis of transmission from cones to hyperpolarizing bipolar cells in the retina of the turtle.

Voltage noise was recorded from centre-hyperpolarizing bipolar cells in the retina of the snapping turtle. The identity of the cells was confirmed by intracellular staining. The variance of the voltage fluctuations of the membrane potential present in the dark was suppressed by up to 30-fold by 100 microns diameter light spot stimuli centred on the cell's receptive field. Such noise reduction is expected when light hyperpolarizes the photoreceptors and reduces the rate of release of transmitter from the terminals. The spectra of the fluctuations were analysed as the sum of two components: (a), a component with power band width limited to below approximately 10 Hz, and (b), a component Sh(f) of the form Sh(f) = Sh(0)/(1 + (f/f0)2)2, with f0 = 27 Hz. The two components were attributed (a) to the noise generated in the cones and transmitted through the synapse to the bipolar cells and (b) to the action of transmitter on the bipolar cell membrane. The component Sh(f) attributed to the action of transmitter on the bipolar cells corresponded to an event approximately 14 ms in duration. The event had a peak amplitude in the range 17.6-223 microV with a mean of 69.5 microV. It is estimated that, in the dark, the number of such events contributing to the noise is about 9200 s-1. It is estimated that each elementary noise event in the cones controls approximately thirty of the transmitter-related events at the synapse. Responses to flashes of darkness applied on steady illumination were analysed by a method of matched filtering. The responses fluctuated in amplitude, and the analysis of this fluctuation suggested an elementary event of approximately the same amplitude as found from the noise analysis. Enlarging the diameter of the stimulus spot to 1500 microns repolarized the bipolar cells with an associated increase in voltage noise. Implications for the synaptic mechanisms of the centre-surround organization are discussed.

Action Potentials↗
PubMed1982

An analysis of voltage noise in rod bipolar cells of the dogfish retina.

1. The power spectral density of voltage noise in depolarizing rod bipolar cells was analysed during darkness and steady illumination. 2. The variance of the voltage fluctuations increased nearly linearly with dim light but was suppressed by bright light. 3. The spectrum in darkness and during illumination could be resolved into two components. One component was attributed to random quantal events arising from spontaneous or light-induced isomerization of rhodopsin in the bipolar cell's rod pool. 4. The second component had a peaked spectrum and was attributed to synaptic noise. 5. A significant fraction of the noise variance in the dark arose from spontaneous thermal isomerization of rhodopsin, with a rate constant of 6 X 10(-12) s-1 or a half-life for rhodopsin of 3700 years at 17 degrees C. 6. The peak amplitude of the single-photon signal in the bipolar cell was about 200 microV, associated with a peak conductance increase of 200 pS. The spectral data suggest that there may be a random delay in the generation of these events. 7. It was concluded that the limitation to single-photon detection in the dark-adapted state may be the rate of spontaneous rhodopsin isomerization. Synaptic noise at the rod-bipolar cell level would not seriously degrade the signal-to-noise ratio.

Animals↗
PubMed1981

Photon-like signals following weak rhodopsin bleaches.

The rod visual system in its dark-adapted state behaves as a near-ideal light detector. Psychophysical studies on the reliability of light detection in man, analysis of the dark noise in rod bipolar cells and observation of photon-like events in rods in the dark suggest that the visual pigment, rhodopsin, is very stable against spontaneous isomerization. When a light which bleaches a small fraction of the rhodopsin is extinguished, the visual threshold may be increased by several orders of magnitude. The eye is then 'light-adapted' and the process (or processes) by which the sensitivity returns constitutes dark adaptation. We report here that bleaching of a small fraction of the rhodopsin produces a prolonged increase in the noise observed in the dark in rod bipolar cells of the dogfish retina. The associated noise events are similar to those produced by the absorption of light quanta and presumably have their origin in the rods which transmit their signals to the bipolar cells. This increased noise after bleaching would decrease the reliability of detection of light quanta and contribute to the elevation of visual threshold.

Animals↗
PubMed1980

Different postsynaptic events in two types of retinal bipolar cell.

The first synapse in the vertebrate visual system is made between the photoreceptors and the biopolar cells. Bioplar cells fall into two distinct classes according to whether the cell hyperpolarizes or depolarizes to small centred spots of light. Most evidence indicates that the light-induced hyperpolarization of the photoreceptprs suppresses transmitter release from the synaptic terminals, and it is probable that the differences between the two bipolar cell classes results from the different actions of the photoreceptor transmitter. In analysing the membrane potential fluctuations in both types of bipolar cell we find that the voltage noise spectra differ. It is to be expected that postsynaptic noise would be composed of the sum of noise generated in and transmitted from the cones and the noise arising from the statistical nature of synaptic transmission. We report here evidence for two such components in the voltage noise spectra recorded from each type of bipolar cell. The differences in the frequency distribution of the presumed transmitter-related components indicates that the transmitter generates events of longer duration in the depolarizing biopolar cells.

Animals↗
PubMed1980

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.2. More than 95% of the bipolar cells sampled were of the type which depolarized to a spot of light. These are termed depolarizing bipolar cells. In most cells, illumination of the surround had little effect on the responses elicited from the central receptive field.3. The mean flash sensitivity of the depolarizing bipolar cells was 270 mV/Rh(**) (where Rh(**) signifies rhodopsin photoisomerization per rod for full field illumination).4. The mean flash sensitivity of horizontal cells under the same conditions was 8 mV/Rh(**). In a limited sample of hyperpolarizing bipolar cells the highest flash sensitivity was 42 mV/Rh(**).5. The high flash sensitivity of the depolarizing bipolar cells indicates a large voltage gain at its synapse with rods. On the assumption of a rod flash sensitivity of 2 mV/Rh(**) the mean gain at the synapse was 135, but for some cells the gain was in excess of 500.6. Responses of depolarizing bipolar cells to dim flashes could be approximated by the impulse response of a 12-16 stage low-pass filter, whereas horizontal cell responses could be fitted by a low-pass filter of six sections. The implied filter at the rod-bipolar cell synapse is tuned to the higher frequency components of rod signals, thereby improving temporal resolution in the rod pathway.7. Depolarizing bipolar cell responses to test flashes are reduced by weak background illumination (less than 0.1 Rh(**)/sec). This desensitization, which would not be expected to affect rod responses, could be explained by a shift in the operating point to a less sensitive region of the intensity-response curve as a result of the large depolarization elicited by the background.8. The results of current injection into the cell in darkness and during the response to light are consistent with the release by rod terminals of a transmitter which closes ionic channels in a conductance path having a reversal potential of - 8 mV, transmitter release being suppressed by light.

Animals↗
PubMed1980

The single-photon signal in rod bipolar cells of the dogfish retina.

1. Responses of depolarizing bipolar cells to dim light flashes were recorded with intracellular micro-electrodes in the dark-adapted retina of the dogfish, Scyliorhinus canicula. 2. Fluctuations in the responses were analysed by a method of matched filtering in order to improve the signal-to-noise ratio. 3. Both the mean and variance of the response amplitude increased linearly with light intensity for intensities not exceeding a mean of 1 photon absorbed per 50 rods. 4. On the assumption that the most significant source of the fluctuation is the quantal absorption of light by the rod outer segments, a single photoisomerization leads to a post-synaptic event of mean size 250 micronV. 5. The mean number of rods in the pool sending signals to a bipolar cell is estimated as 1600. Individual rod pools are 90-330 micrometer in diameter on the retinal surface. 6. It is estimated that the conductance of 1 divided by 400 of the total number of light-modulated ionic channels in the bipolar cell is increased by a single photon acting within its rod pool. 7. In the absence of a light stimulus, the residual noise in the output of a matched filter can be interpreted as due mainly to spontaneous isomerization of rhodopsin in the rods and behaves as the 'dark light' postulated to limit detection at absolute threshold.

Animals↗