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Biomedical subjects

J F Ashmore

Publications and source records attributed to J F Ashmore.

At least 37 records · Page 2Linked to original sources
PubMed1995

Action of salicylate on membrane capacitance of outer hair cells from the guinea-pig cochlea.

1. The effect of salicylate on membrane capacitance and intracellular pH has been measured in isolated outer hair cells (OHCs) during whole cell recording. Cell membrane capacitance was measured using a lock-in amplifier technique. 2. Salicylate applied in the bath reduced the fast charge movement, equivalent to a voltage-dependent membrane capacitance, present in OHCs. Simultaneous measurement of membrane capacitance and voltage-driven cell length changes showed that salicylate reduced both together. 3. A small effect of salicylate on outward currents at 0 mV was observed. Sodium salicylate (5 mM) reduced the currents by 19% and another weak acid, sodium butyrate (10 mM), reduced outward currents in OHCs by 15%. 4. The ratiometric dye 2,7-bis(2-carboxymethyl)-5,6-carboxyfluorescein (BCECF) was used to measure pHi changes in OHCs during weak acid exposure. Membrane capacitance and pHi were measured simultaneously in OHCs exposed first to 10 mM sodium butyrate and then to 5 mM sodium salicylate. Although both compounds produced a similar reduction in pHi, butyrate decreased the resting capacitance from a mean resting capacitance of 35 pF (at -30 mV) by 5.4 +/- 2.1 pF, whereas salicylate decreased it by 15.7 +/- 2.3 pF (n = 4). 5. Exposure of OHCs to 10 mM sodium benzoate, an amphiphilic anion, reduced resting membrane capacitance at -30 mV by 9.2 +/- 3.2 pF (n = 3). Outward currents, measured at 0 mV, were reduced by 0.25 +/- 0.05 nA during benzoate application, comparable with the effect of salicylate. 6. Capacitance was measured during slow bath application of salicylate. The resulting dose-capacitance curve had a Hill coefficient of 3.40 +/- 0.85 (n = 4) and a half-maximal dose of 3.95 +/- 0.34 mM. The dose-capacitance curve was not significantly voltage dependent. 7. Salicylate had no detectable effect on the resting capacitance of Deiters' cells, a non-sensory cell type of the organ of Corti. 8. It is concluded that many of the described effects of salicylate on hearing may arise from the partitioning of the salicylate molecule into the membrane of the OHC and consequent inhibition of OHC motility.

Animals↗
PubMed1995

Patch clamped responses from outer hair cells in the intact adult organ of Corti.

Outer hair cells (OHCs) from the mammalian cochlea act as both sensory cells and motor cells. We report here whole-cell tight seal recordings of OHC activity in their natural embedding tissue, the intact organ of Corti, using a temporal bone preparation. The mean cell resting potential, -76 +/- 4 mV (n = 19) and input conductance (10 +/- 3 nS at -70 mV) of third turn hair cells were significantly lower than have been found in isolated cells. Two main K+ currents in the cell were identified. One current, activated positive to -100 mV, was reduced by 5 mM BaCl2. The other current, activated above -40 mV, was reduced by 100 microM 4-aminopyridine (4-AP) and by 30 mM tetraethylammonium (TEA). Both of these currents have been also identified in recordings reported from isolated cells. On stepping to different membrane potentials, cells imaged in the organ of Corti changed length by an amount large enough to cause visible distortions in neighbouring cells. By quantifying such distortions we estimate that the forces generated by OHCs can account for the enhanced response to sound required by the cochlear amplifier.

Animals↗
PubMed1995

A laser interferometer for sub-nanometre measurements in the cochlea.

A modification of a light microscope is described here which allows measurement of nanometre movements along the optical axis of the microscope. The light path is reflected off 10-microns-diametre small glass beads which are individually imaged with the microscope objective. The interferometer is under computer control to allow it to remain in quadrature and so maximise sensitivity. The algorithm is described. Although the techniques are applied to detection of movements of the cochlear partition, they can be used to measure sub-micron movements of any reflecting structures accessible to microscopy.

Animals↗
PubMed1995

Transduction and adaptation in sensory receptor cells.

Sensory transduction shares common features in widely different sensory modalities. The purpose of this article is to examine the similarities and differences in the underlying mechanisms of transduction in the sensory receptor cells for vision, olfaction, and hearing. One of the major differences between the systems relates to the nature of the stimulus. In both the visual and olfactory systems a quantal mechanism of detection is possible, because the absorption of a photon or the binding of an odorant molecule provides an energy change significantly greater than the thermal noise in the receptor molecule. In hearing, on the other hand, the energy of a phonon is far lower, and detection occurs by a "classical" mechanism. For vertebrate photoreceptors and olfactory receptor cells, sensory transduction employs a G protein cascade that is remarkably similar in the two cases, and that is closely homologous to other G protein signaling cascades. For auditory and vestibular hair cells, transduction operates via a mechanism of direct coupling of the stimulus to ion channels, in a manner reminiscent of the direct gating of post-synaptic ion channels in various synaptic mechanisms. The three classes of sensory receptor cell share similarities in their mechanisms of adaptation, and it appears in each case that cytoplasmic calcium concentration plays a major role in adaptation.

Adaptation, Physiological↗
PubMed1994

Charge displacement induced by rapid stretch in the basolateral membrane of the guinea-pig outer hair cell.

The properties of the basolateral membrane of cochlear outer hair cells were studied under whole-cell patch clamp to measure currents and capacitance changes associated with mechanical deformation. Stretching the membrane of outer hair cells along the cell axis generated a transient inward current, and subsequent relaxation of the membrane produced a similar transient outward current. These mechanically activated currents were velocity dependent with a mean sensitivity of 29 pA s mm-1. Unlike ionic currents, these currents did not reverse, but reached a peak magnitude at -33 mV. Stretching the cell also resulted in a measurable capacitance decrease of 0.3-1.1 pF microns-1. These results suggest that membrane stretch can induce a rapid charge movement resulting from the reversal of the electromechanical transduction process in outer hair cells.

Animals↗
PubMed1994

Hair cell based amplification in the cochlea.

Recently, several reports have provided data about the way in which outer hair cells amplify sound within the cochlea. These include new measurements of hair cell forces in the cochlea and in isolated hair cells. In addition, theoretical advances that synthesize such experiments and show how hair cells might tune up the cochlear response sound promising.

Animals↗
PubMed1993

Reverse transduction measured in the isolated cochlea by laser Michelson interferometry.

It is thought that the sensitivity of mammalian hearing depends on amplification of the incoming sound within the cochlea by a select population of sensory cells, the outer hair cells. It has been suggested that these cells sense displacements and feedback forces which enhance the basilar membrane motion by reducing the inherent damping of the cochlear partition. In support of this hypothesis, outer hair cells show membrane-potential-induced length changes at acoustic rates. This process has been termed 'reverse transduction'. For amplification, the forces should be large enough to move the basilar membrane. Using a displacement-sensitive interferometer, we tested this hypothesis in an isolated cochlea while stimulating the outer hair cells with current passed across the partition. We show here that the cochlear partition distorts under the action of electrically driven hair cell length changes and produces place-specific vibration of the basilar membrane of a magnitude comparable to that observed near auditory threshold (about 1 nm). Such measurements supply direct evidence that cochlear amplification arises from the properties of the outer hair cell population.

Animals↗
PubMed1993

Effects of extracellular ATP on hair cells isolated from the guinea-pig semicircular canals.

Externally applied ATP (100 microM) induced membrane currents in type I and type II vestibular hair cells enzymatically isolated from guinea-pig semicircular canals. In whole-cell voltage-clamp and with 140 mM K+ in the pipette solution, ATP evoked an inwardly directed current in 58% of the cells when held at potentials below -40 mV. In the remainder, external ATP produced an outward current. After block of the K currents, an inward current activated by ATP was revealed at -50 mV. Intracellular Ca2+ levels were monitored using the Ca2+ indicator Fura-2 and were found to rise in both hair cell types in response to ATP. These results strongly suggest that ATP directly controls the entry of Ca2+ into crista hair cells which can then further modulate K+ currents.

Adenosine Triphosphate↗
PubMed1993

GABAB-mediated modulation of ionic conductances in type I hair cells isolated from guinea-pig semicircular canals.

Mammalian vestibular type I hair cells (VIHCs) are innervated by an afferent synaptic calyx which contains vesicles and is immunoreactive for GABA. We describe here the effects of GABA on electrophysiological properties and on cytosolic free-calcium levels ([Ca2+]i) of VIHCs isolated from guinea-pig ampullae. Whole-cell tight-seal macroscopic currents recorded from VIHCs showed that 100 microM GABA induced a decrease in the outward currents elicited by depolarizing membrane potentials. These are known to comprise potassium calcium-dependent currents. This effect was mimicked by baclofen, a GABAB agonist, and was not affected by picrotoxin, a GABAA antagonist. GABA also induced an increase in the inward current elicited at hyperpolarized membrane potentials in 50% of the tested cells. Single channel recording in cell-attached patches revealed that externally applied GABA produced a decrease and an increase in the open probability of 170 pS and 45 pS and of 15 pS channels, respectively. In imaging experiments using the dye Fura-2 to measure [Ca2+]i, the only reversible modulation of [Ca2+]i observed in response to GABA application was a decrease. These results demonstrate a modulation of calcium and potassium conductances by GABA, via GABAB receptors, in guinea-pig VIHCs.

Animals↗
PubMed1992

Localization of cholinergic and purinergic receptors on outer hair cells isolated from the guinea-pig cochlea.

Acetylcholine (ACh) and adenosine 5'-triphosphate (ATP) are shown to act in opposing fashion on guinea-pig cochlear outer hair cells (OHCS) via receptors localized within different fluid compartments of the organ of Corti. The cholinergic (efferent) receptors localized at the basal (perilymphatic) region of these cells activated a rapidly desensitizing hyperpolarizing K+ current. In contrast, purinergic (ATP) receptors were localized at the apical (endolymphatic) surface of OHCS and activated a depolarizing nonselective cation current which exhibited inward rectification and lacked desensitization. Localization of the receptors was determined by using whole-cell patch-clamp, by recording onset latencies and response amplitudes to pulses of either ACh or ATP pressure-applied at selected sites along the length of isolated OHCS. Under voltage-clamp at -60 mV, the largest ACh-induced (outward) currents were recorded when ACh was directed at the basal region of the cells. Conversely, the maximum (inward) ATP currents were obtained when ATP was directed toward the apical surface of these cells. Onset latencies increased rapidly from a minimum of approximately 10 ms for either ACh or ATP as the drug pipette was moved away from these optimal sites. The ATP response was antagonized by amiloride in a dose-dependent manner with a KD of approximately 400 microM. The localization of P2-type purinoceptors to the endolymphatic surface of OHCS suggests that ATP mediates a humoral modulation of the mechano-electrical transduction process.

Acetylcholine↗
PubMed1992

Ionic currents of outer hair cells isolated from the guinea-pig cochlea.

1. Whole-cell currents were measured in outer hair cells isolated from each turn of the organ of Corti of the guinea-pig. 2. The slope input conductances at -70 mV of the cells ranged from 3.6 to 51 nS depending on the length of the cell. Shorter cells from the basal turns of the cochlea had the highest values. The membrane time constant of the cells varied from 3 to 0.2 ms from the apex to the base. 3. Irrespective of the position of the cells along the cochlea, three distinct currents were found. Each type of current was found in approximately the same proportion in all cells. 4. An outward K+ current was present which activated at potentials more positive than -35 mV. The current was sensitive to tetraethylammonium (30 mM), quinidine (100 microM) and nifedipine (50 microM). It could be removed by replacing external Ca2+ with Ba2+ or Mg2+. The current was also removed by substituting Nai+ or Csi+ for Ki+ pipette solution. This outwardly rectifying current appears similar to the calcium-activated K+ current described in other hair cells. 5. The main current present at membrane potentials from -90 mV to -50 mV was a second voltage-activated K+ current. It was 50% activated at -80 mV, and relaxed with a time constant of 20-40 ms on hyperpolarization to -120 mV. Near rest the kinetics were essentially time-dependent , but depended upon the external K+ concentration. The current was blocked by 5 mM external Cs+. 6. This current was highly selective for K+. Measured from reversal of the tail currents, the permeability ratio PK:PNa was approximately 30:1. Depolarization of the cell, presumed to lead to an elevation of intracellular calcium, produced a prolonged activation of the current. 7. A third current found in the cells was a cation current. By external ion replacement, the selectivity sequence was determined to be Ca2+ greater than Na+ approximately equal to K+ greater than choline+ greater than NMDG+ (respective permeabilities relative to Na: 2.9, 1.0, 0.99, 0.63 and 0.37). This current was reduced by external Ba2+ (3 mM) and by nifedipine (50 microM). The activation of this current appeared to depend upon raised levels of Cai2+. 8. These currents account for reported in vivo properties of cochlear outer hair cells as cells permeable to potassium at large negative resting potentials. The consequences for sound detection in the cochlea are briefly discussed.

Animals↗
PubMed1991

Direct measurement of the action of acetylcholine on isolated outer hair cells of the guinea pig cochlea.

Acetylcholine has long been thought to be the neurotransmitter of the cochlear efferent system in mammals although the evidence is largely indirect. By using whole-cell recordings from isolated outer hair cells, we show that acetylcholine activates a large rapidly desensitizing outward potassium current. This corresponds to hyperpolarization of the membrane potential from rest. The half maximal dose for acetylcholine was 13.5 microM with a cooperativity of 2. The response was not due to a conventional muscarinic action of acetylcholine for it was not blocked by 0.1 microM atropine and muscarinic antagonists but it could be blocked by 0.1 microM curare, suggesting that it shared many properties of a nicotinic receptor. It was, however, inhibited by 10 microM strychnine. The potassium current activated by acetylcholine required external calcium and was characterized by a significant delay at room temperature. This points to the involvement of a second messenger system, possibly calcium itself.

Acetylcholine↗
PubMed1991

Ionic currents in isolated vestibular hair cells from the guinea-pig crista ampullaris.

Ionic currents have been recorded under whole cell patch clamp in cells isolated from the guinea-pig vestibular system. Type I and type II cells were separately identified. Type II cells were further classified as short (less than 15 microns in length) or tall (greater than 15 microns). Under whole cell voltage clamp, cells showed an outward current which activated at potentials above about -50 mV, and tail currents which reversed near the potassium equilibrium potential. The outward current was reduced in the presence of external 10 mM tetraethylammonium or cadmium ions and when calcium was removed from the external medium. A small cadmium-sensitive transient inward current, a putative calcium current, was observed in cells loaded with caesium from the patch pipette. In 27 out of 64 cells a component of the recorded outward current inactivated. Such current components were most common in tall type II cells. This inactivating component was blocked by 4-aminopyridine and removed by depolarizing prepulses consistent with it being an A-type potassium current. Type I cells, on the other hand, showed mainly a non-inactivating outward current which slowly relaxed on repolarization to resting potentials. When membrane potentials were measured under current clamp, injections of less than 100 pA produced a single, highly damped transient followed by a plateau in Type II cells. No such transient was present in Type I cells. There is thus little evidence for an electrical resonance in these cells.

4-Aminopyridine↗
PubMed1990

A cytoskeletal spring for the control of cell shape in outer hair cells isolated from the guinea pig cochlea.

A two-dimensional cortical cytoskeletal lattice associated with the lateral plasma membranes of mammalian outer hair cells maintains cell shape and provides a restoring force to oppose active changes in cell length. The lattice is composed of two morphologically distinct filaments which are arranged to reinforce the cell circumferentially whilst allowing limited changes in cell length and diameter. This function can only be fulfilled if intracellular pressure is high enough to put the lattice under tension.

Animals↗