Live imaging the phagocytic activity of inner ear supporting cells in response to hair cell death.

Hearing loss and balance disorders affect millions of people worldwide. Sensory transduction in the inner ear requires both mechanosensory hair cells (HCs) and surrounding glia-like supporting cells (SCs). HCs are susceptible to death from aging, noise overexposure, and treatment with therapeutic drugs that have ototoxic side effects; these ototoxic drugs include the aminoglycoside antibiotics and the antineoplastic drug cisplatin. Although both classes of drugs are known to kill HCs, their effects on SCs are less well understood. Recent data indicate that SCs sense and respond to HC stress, and that their responses can influence HC death, survival, and phagocytosis. These responses to HC stress and death are critical to the health of the inner ear. Here we have used live confocal imaging of the adult mouse utricle, to examine the SC responses to HC death caused by aminoglycosides or cisplatin. Our data indicate that when HCs are killed by aminoglycosides, SCs efficiently remove HC corpses from the sensory epithelium in a process that includes constricting the apical portion of the HC after loss of membrane integrity. SCs then form a phagosome, which can completely engulf the remaining HC body, a phenomenon not previously reported in mammals. In contrast, cisplatin treatment results in accumulation of dead HCs in the sensory epithelium, accompanied by an increase in SC death. The surviving SCs constrict fewer HCs and display impaired phagocytosis. These data are supported by in vivo experiments, in which cochlear SCs show reduced capacity for scar formation in cisplatin-treated mice compared with those treated with aminoglycosides. Together, these data point to a broader defect in the ability of the cisplatin-treated SCs, to preserve tissue health in the mature mammalian inner ear.

FM1-43 dye behaves as a permeant blocker of the hair-cell mechanotransducer channel.

Hair cells in mouse cochlear cultures are selectively labeled by brief exposure to FM1-43, a styryl dye used to study endocytosis and exocytosis. Real-time confocal microscopy indicates that dye entry is rapid and via the apical surface. Cooling to 4 degrees C and high extracellular calcium both reduce dye loading. Pretreatment with EGTA, a condition that breaks tip links and prevents mechanotransducer channel gating, abolishes subsequent dye loading in the presence of calcium. Dye loading recovers after calcium chelation with a time course similar to that described for tip-link regeneration. Myo7a mutant hair cells, which can transduce but have all mechanotransducer channels normally closed at rest, do not label with FM1-43 unless the bundles are stimulated by large excitatory stimuli. Extracellular perfusion of FM1-43 reversibly blocks mechanotransduction with half-blocking concentrations in the low micromolar range. The block is reduced by high extracellular calcium and is voltage dependent, decreasing at extreme positive and negative potentials, indicating that FM1-43 behaves as a permeant blocker of the mechanotransducer channel. The time course for the relief of block after voltage steps to extreme potentials further suggests that FM1-43 competes with other cations for binding sites within the pore of the channel. FM1-43 does not block the transducer channel from the intracellular side at concentrations that would cause complete block when applied extracellularly. Calcium chelation and FM1-43 both reduce the ototoxic effects of the aminoglycoside antibiotic neomycin sulfate, suggesting that FM1-43 and aminoglycosides enter hair cells via the same pathway.

Solitary hair cells are distributed throughout the extramacular epithelium in the bullfrog's saccule.

The frog inner ear contains eight sensory organs that provide sensitivities to auditory, vestibular, and ground-borne vibrational stimuli. The saccule in bullfrogs is responsible for detecting ground- and airborne vibrations and is used for studies of hair cell physiology, development, and regeneration. Based on hair bundle morphology, a number of hair cell types have been defined in this organ. Using immunocytochemistry, vital labeling, and electron microscopy, we have characterized a new hair cell type in the bullfrog saccule. A monoclonal antibody that is specific to hair cells revealed that a population of solitary hair cells exists outside the sensory macula in what was previously thought to be nonsensory epithelium. We call these extramacular hair cells. There are 80-100 extramacular hair cells in both tadpole and adult saccules, which extend up to 1 mm from the edge of the sensory macula. The extramacular hair cells have spherical cell bodies and small apical surfaces. Even in adults, the hair bundles of the extramacular cells appear immature, with a long kinocilium (6-9 microm) and short stereocilia (0.5-2 microm). At least 90% of extramacular hair cells are likely to be innervated as demonstrated by labeling of nerve fibers with an antineurofilament antibody. The extramacular hair cells may differentiate in regionsjust beyond the edge of the macula at an early stage in development and then be pushed out via the interstitial growth of the epithelium that surrounds the macula. It is also possible that they may be produced from cell divisions in the extramacular epithelium that has not been considered capable of giving rise to hair cells.

An intrinsic frequency limit to the cochlear amplifier.

Hearing in mammals depends on a feedback process within the inner ear termed the 'cochlear amplifier'. The essential components of this amplifier are sensorimotor cells, the outer hair cells, which transduce motion of the basilar membrane induced by sound and generate forces to cancel the viscous damping of the cochlear partition. Outer hair cells alter the passive mechanics of the cochlea, enhancing both the sensitivity and the frequency selectivity of the auditory system. The molecular basis of the mechanism is thought to be a voltage-sensitive 'motor' protein, as yet unidentified, embedded in the basolateral membrane of the outer hair cell. The cochlear amplifier operates up to at least 22 kHz, but by measuring both the charge and mechanical movements associated with the motor in isolated membrane patches under voltage clamp, we show here that the limiting frequency at which the motor operates lies near 25 kHz. This value therefore sets an upper limit to the range of hearing in mammalian cochleas using this mechanism. The fast charge movement, arising from charge displacement within the presumed motor molecule, further suggests that the protein is more likely to be related to a transporter than to a modified ion channel.

The outer hair cell motor in membrane patches.

We have used patch-clamp techniques to record the charge movement associated with motility in patches of basolateral membrane from isolated outer hair cells. Charge movement has been measured from the voltage-dependent capacitance. Using 3 to 4 Momega pipettes with tip diameters of 3 micron the measured maximum voltage-dependent capacitance was 56 +/- 6 fF at -36 mV when the resting membrane potential was -20 mV. The calculated total charge movement was 5.6 +/- 0.6 fC (n = 13) and the inferred density of univalent motor elements was 8400/micron2. Negative pressure (applied via the pipette) increased membrane tension and shifted the capacitance peak to depolarised potentials. Under conditions of isotropic membrane stress there was no change in the peak measured capacitance in contrast to that measured in previous whole-cell recordings.

Decentering therapy: textual analysis of a narrative therapy session.

Postmodern models of therapy stress the participation of the clinician in a nonhierarchical, non-objectifying role, and highlight the therapist's embeddedness in the same processes of social construction as are the individual and the family. While much theory has been published in recent years, the actual conduct of a therapy session derived from the premises of postmodernism remains unclear. We investigated how a postmodern therapist manages talk in an actual session. We used textual analysis to examine a couples therapy session conducted by a prominent narrative therapist. Analysis of the talk led to descriptions of the couple's and therapist's agenda, and their interaction. The therapist's agenda is described in terms of "decentering" both the local unfolding narrative and its embeddedness in larger cultural stories. Five conversational practices: matching/self-disclosure, reciprocal editing, turn management to deobjectify, expansion questions, and reversals are examined. These practices inform the deployment of a decentering agenda in this specific text.

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.

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.

Geologic storage of radioactive waste: field studies in Sweden.

Access to a granitic rock mass in an iron ore mine in Sweden provided a unique opportunity for underground experiments related to the geologic disposal of radioactive waste. These field tests demonstrated the importance of hydrogeology and the difficulties in predicting the thermomechanical behavior of fractured granitic rocks. To characterize a site fully, measurements made from the surface must be supplemented by extensive subsurface measurements and experiments. Much effort is needed at this stage to generate the technology required for the development of waste repositories.