Rise Time Reduction of Thermal Actuators Operated in Air and Water through Optimized Pre-Shaped Open-Loop Driving.

Electrothermal actuators have many advantages compared to other actuators used in Micro-Electro-Mechanical Systems (MEMS). They are simple to design, easy to fabricate and provide large displacements at low voltages. Low voltages enable less stringent passivation requirements for operation in liquid. Despite these advantages, thermal actuation is typically limited to a few kHz bandwidth when using step inputs due to its intrinsic thermal time constant. However, the use of pre-shaped input signals offers a route for reducing the rise time of these actuators by orders of magnitude. We started with an electrothermally actuated cantilever having an initial 10-90% rise time of 85 µs in air and 234 µs in water for a standard open-loop step input. We experimentally characterized the linearity and frequency response of the cantilever when operated in air and water, allowing us to obtain transfer functions for the two cases. We used these transfer functions, along with functions describing desired reduced rise-time system responses, to numerically simulate the required input signals. Using these pre-shaped input signals, we improved the open-loop 10-90% rise time from 85 µs to 3 µs in air and from 234 µs to 5 µs in water, an improvement by a factor of 28 and 47, respectively. Using this simple control strategy for MEMS electrothermal actuators makes them an attractive alternative to other high speed micromechanical actuators such as piezoelectric stacks or electrostatic comb structures which are more complex to design, fabricate, or operate.

Response properties from turtle auditory hair cell afferent fibers suggest spike generation is driven by synchronized release both between and within synapses.

Inner ear hair cell afferent fiber synapses are capable of transferring information at high rates for long periods of time with extraordinary fidelity. As at other sensory synapses, hair cells rely on graded receptor potentials and unique vesicle trafficking and release properties of ribbon synapses to relay intensity information. Postsynaptic recordings from afferent fibers of the turtle auditory papilla identified excitatory postsynaptic currents (EPSCs) that were fast AMPA receptor-based responses with rapid onset and decay times. EPSCs varied in amplitude by ≈ 15× per fiber, with kinetics that showed a tendency to slow at larger amplitudes. Complex EPSCs were produced by temporal summation of single events, likely across synapses. Complex EPSCs were more efficient at generating action potentials than single EPSCs. Potassium-evoked release increased the frequency of EPSCs, in particular complex events, but did not increase EPSC amplitudes. Temporal summation of EPSCs across synapses may underlie action potential generation at these synapses. Broad amplitude histograms were probed for mechanisms of multivesicular release with reduced external Ca(2+) or the introduction of Cd(2+) or Sr(2+) to uncouple release. The results are consistent with broad amplitude histograms being generated by a combination of the variability in synaptic vesicle size and coordinated release of these vesicles. It is posited that multivesicular release plays less of a role in multisynaptic ribbon synapses than in single synaptic afferent fibers.

Permeation properties of the hair cell mechanotransducer channel provide insight into its molecular structure.

Mechanoelectric transducer (MET) channels, located near stereocilia tips, are opened by deflecting the hair bundle of sensory hair cells. Defects in this process result in deafness. Despite this critical function, the molecular identity of MET channels remains a mystery. Inherent channel properties, particularly those associated with permeation, provide the backbone for the molecular identification of ion channels. Here, a novel channel rectification mechanism is identified, resulting in a reduced pore size at positive potentials. The apparent difference in pore dimensions results from Ca(2+) binding within the pore, occluding permeation. Driving force for permeation at hyperpolarized potentials is increased because Ca(2+) can more easily be removed from binding within the pore due to the presence of an electronegative external vestibule that dehydrates and concentrates permeating ions. Alterations in Ca(2+) binding may underlie tonotopic and Ca(2+)-dependent variations in channel conductance. This Ca(2+)-dependent rectification provides targets for identifying the molecular components of the MET channel.

Mechanisms of aminoglycoside ototoxicity and targets of hair cell protection.

Aminoglycosides are commonly prescribed antibiotics with deleterious side effects to the inner ear. Due to their popular application as a result of their potent antimicrobial activities, many efforts have been undertaken to prevent aminoglycoside ototoxicity. Over the years, understanding of the antimicrobial as well as ototoxic mechanisms of aminoglycosides has increased. These mechanisms are reviewed in regard to established and potential future targets of hair cell protection.

Mechano-electrical transduction: new insights into old ideas.

The gating-spring theory of hair cell mechanotransduction channel activation was first postulated over twenty years ago. The basic tenets of this hypothesis have been reaffirmed in hair cells from both auditory and vestibular systems and across species. In fact, the basic findings have been reproduced in every hair cell type tested. A great deal of information regarding the structural, mechanical, molecular and biophysical properties of the sensory hair bundle and the mechanotransducer channel has accumulated over the past twenty years. The goal of this review is to investigate new data, using the gating spring hypothesis as the framework for discussion. Mechanisms of channel gating are presented in reference to the need for a molecular gating spring or for tethering to the intra- or extracellular compartments. Dynamics of the sensory hair bundle and the presence of motor proteins are discussed in reference to passive contributions of the hair bundle to gating compliance. And finally, the molecular identity of the channel is discussed in reference to known intrinsic properties of the native transducer channel.

Auditory hair cell-afferent fiber synapses are specialized to operate at their best frequencies.

Auditory afferent fiber activity is driven by high-fidelity information transfer from the sensory hair cell. Presynaptic specializations, posited to maintain fidelity, are investigated at synapses with characteristic frequencies of 120 Hz and 320 Hz. Morphological data indicate that high-frequency cells have more synapses and higher vesicle density near dense bodies (DBs). Tracking vesicular release via capacitance changes identified three overlapping kinetic components of release corresponding to morphologically identified vesicle pools. High-frequency cells released faster; however, when normalized to release site number, low-frequency cells released faster, likely due to a greater Ca2+ load per synapse. The Ca(2+)-dependence of release was nonsaturating and independent of frequency, suggesting that release, not refilling, was rate limiting. A model of release derived from vesicle equilibration between morphologically defined pools reproduced the capacitance data, supporting a critical role in vesicle trafficking for DBs. The model suggests that presynaptic specializations enable synapses to operate most efficiently at their characteristic frequencies.

Probing the pore of the auditory hair cell mechanotransducer channel in turtle.

Hair cell mechano-electric transducer (MET) channels play a pivotal role in auditory and vestibular signal detection, yet few data exist regarding their molecular nature. Present work characterizes the MET channel pore, a region whose properties are thought to be intrinsically determined. Two approaches were used. First, the channel was probed with antagonists of candidate channel subtypes including: cyclic nucleotide-gated channels, transient receptor potential channels and gap-junctional channels. Eight new antagonists were identified. Most of the effective antagonists had a partially charged amine group predicted to penetrate the channel pore, antagonizing current flow, while the remainder of the molecule prevented further permeation of the compound through the pore. This blocking mechanism was tested using curare to demonstrate the open channel nature of the block and by identifying methylene blue as a permeant channel blocker. The second approach estimated dimensions of the channel pore with simple amine compounds. The narrowest diameter of the pore was calculated as 12.5 +/- 0.8 A and the location of a binding site approximately 45% of the way through the membrane electric field was calculated. Channel length was estimated as approximately 31 A and the width of the pore mouth at < 17 A. Each effective antagonist had a minimal diameter, measured about the penetrating amine, of less than the pore diameter, with a direct correlation between IC(50) and minimal diameter. The IC(50) was also directly related to the length of the amine side chains, further validating the proposed pore blocking mechanism. Data provided by these two approaches support a hypothesis regarding channel permeation and block that incorporates molecular dimensions and ion interactions within the pore.

Mechano-electrical transduction in the turtle utricle.

Two classes of mechano-sensory hair cell are present in the vestibular system of mammals, birds and reptiles. Type I hair cells are bottle-shaped and make synaptic contact with afferent calyx terminals. Type II hair cells are cylindrical and contact small bouton afferent terminals. Voltage-dependent basolateral currents have been found to differ between the two cell types and these properties are believed to contribute to the shaping of primary afferent responses. Type I hair cells have a low input resistance, which may be related to the low gain of calyx afferents. In the turtle utricle, type I vestibular hair cells are found only in a narrow band of the sensory epithelium called the striola, whereas type II hair cells are found in both striolar and extrastriolar regions. We have made whole cell patch clamp recordings from type I hair cells, type II hair cells and calyx fibers in order to better understand the processing of vestibular signals. Here we describe responses of hair cells to hair bundle displacement with a stiff glass probe. Mechano-electrical transduction (MET) currents were largest in type I hair cells, where the mean peak amplitude was approximately 500 pA. MET currents in all hair cells showed rapid and slow adaptation.

Biophysical and pharmacological characterization of voltage-gated calcium currents in turtle auditory hair cells.

Hair cell calcium channels regulate membrane excitability and control synaptic transmission. The present investigations focused on determining whether calcium channels vary between hair cells of different characteristic frequencies or if multiple channel types exist within a hair cell, each serving a different function. To this end, turtle auditory hair cells from high- (317 +/- 27 Hz) and low-frequency (115 +/- 6 Hz) positions were voltage clamped using the whole-cell recording technique, and calcium currents were characterized based on activation, inactivation and pharmacological properties. Pharmacological sensitivity to dihydropyridines (nimodipine, Bay K 8644), benzothiazepines (diltiazem) and acetonitrile derivatives (verapamil, D600) and the insensitivity to non-L-type calcium channel antagonists support the conclusion that only L-type calcium channels were present. Fast activation rise times (< 0.5 ms), hyperpolarized half-activation potentials and a relative insensitivity to nimodipine suggest the channels were of the alpha1D (CaV1.3) variety. Although no pharmacological differences were found between calcium currents obtained from high- and low-frequency cells, low-frequency cells activated slightly faster and at hyperpolarized potentials, with half-activating voltages of -43 +/- 1 mV compared to -35 +/- 1 mV. Inactivation was observed in both high- and low-frequency cells. The time course of inactivation required three time constants for a fit. Long depolarizations could result in complete inactivation. The voltage of half-inactivation was -40 +/- 2 mV for high-frequency cells and -46 +/- 2 mV for low-frequency cells. Calcium channel inactivation did not significantly alter hair cell electrical resonant properties elicited from protocols where the membrane potential was hyperpolarized or depolarized prior to characterizing the resonance. A bell-shaped voltage dependence and modest sensitivities to intracellular calcium chelators and external barium ions suggest that inactivation was calcium dependent.

Mechanisms of active hair bundle motion in auditory hair cells.

Sound stimuli vibrate the hair bundles on auditory hair cells, but the resulting motion attributable to the mechanical stimulus may be modified by forces intrinsic to the bundle, which drive it actively. One category of active hair bundle motion has properties similar to fast adaptation of the mechanotransducer channels and is explicable if gating of the channels contributes significantly to the mechanics of the hair bundle. To explore this mechanism, we measured hair bundle compliance in turtle auditory hair cells under different conditions that alter the activation range of the channel. Force-displacement relationships were nonlinear, possessing a maximum slope compliance when approximately one-half of the transducer channels were open. When the external calcium concentration was reduced from 2.8 to 0.25 mm, the position of maximum compliance was shifted negative, reflecting a comparable shift in the transducer channel activation curve. Assuming that the nonlinearity represents the compliance attributable to channel gating, a single-channel gating force of 0.25 pN was calculated. By comparing bundle displacements with depolarization with and without an attached flexible fiber, the force contributed by each channel was independently estimated as 0.47 pN. These results are consistent with fast active bundle movements resulting from changes in mechanotransducer channel gating. However, several observations revealed additional components of hair bundle motion, with slower kinetics and opposite polarity to the fast movement but also linked to transducer adaptation. This finding argues for multiple mechanisms for controlling hair bundle position in auditory hair cells.

Clues to the cochlear amplifier from the turtle ear.

Sound stimuli are detected in the cochlea by vibration of hair bundles on sensory hair cells, which activates mechanotransducer ion channels and generates an electrical signal. Remarkably, the process can also work in reverse with additional force being produced by the ion channels as they open and close, evoking active movements of the hair bundle. These movements could supplement the energy of the sound stimuli but to be effective they would need to be very fast. New measurements in the turtle ear have shown that such active bundle movements occur with delays of less than a millisecond, and are triggered by the entry of Ca(2+) into the cell via the mechanotransducer channel. Furthermore, their speed depends on the frequency to which the hair cell is most sensitive, suggesting that such movements could be important in cochlear amplification and frequency discrimination.

Active hair bundle motion linked to fast transducer adaptation in auditory hair cells.

During transduction in auditory hair cells, hair bundle deflection opens mechanotransducer channels that subsequently reclose or adapt to maintained stimuli, a major component of the adaptation occurring on a submillisecond time scale. Using a photodiode imaging technique, we measured hair bundle motion in voltage-clamped turtle hair cells to search for a mechanical correlate of fast adaptation. Excitatory force steps imposed by a flexible glass fiber attached to the bundle caused an initial movement toward the kinocilium, followed by a fast recoil equivalent to bundle stiffening. The recoil had a time course identical to adaptation of the transducer current, and like adaptation, was most prominent for small stimuli, was slowed by reducing extracellular calcium, and varied with hair cell resonant frequency. In free-standing hair bundles, depolarizations positive to 0 mV evoked an outward current attributable to opening of transducer channels, which was accompanied by a sustained bundle deflection toward the kinocilium. Both processes were sensitive to external calcium concentration and were abolished by blocking the transducer channels with dihydrostreptomycin. The similarity in properties of fast adaptation and the associated bundle motion indicates the operation of a rapid calcium-sensitive force generator linked to the gating of the transducer channels. This force generator may permit stimulus amplification during transduction in auditory hair cells.

Tonotopic variations of calcium signalling in turtle auditory hair cells.

Turtle cochlear hair cells are electrically tuned by a voltage-dependent Ca2+ current and a Ca2+-dependent K+ current (IBK(Ca)). The effects of intracellular calcium buffering on electrical tuning were studied in hair cells at apical and basal cochlear locations tuned to 100 and 300 Hz, respectively. Increasing the intracellular BAPTA concentration changed the hair cell's resonant frequency little, but optimized tuning at more depolarized membrane potentials due to a positive shift in the half-activation voltage (V ) of the IBK(Ca). The shift in V depended similarly on BAPTA concentration in basal and apical hair cells despite a 2. 4-fold difference in the size of the Ca2+ current at the two positions. The Ca2+ current amplitude increased exponentially with distance along the cochlea. Comparison of V values and tuning properties using different BAPTA concentrations with values measured in perforated-patch recordings gave the endogenous calcium buffer as equivalent to 0.21 mM BAPTA in low-frequency cells, and 0.46 mM BAPTA in high-frequency cells. High conductance Ca2+-activated K+ (BKCa) channels recorded in inside-out membrane patches were 2-fold less Ca2+ sensitive in high-frequency than in low-frequency cells. Confocal Ca2+ imaging using the fluorescent indicator Calcium Green-1 revealed about twice as many hotspots of Ca2+ entry during depolarization in high-frequency compared to low-frequency hair cells. We suggest that each BKCa channel is gated by Ca2+ entry through a few nearby Ca2+ channels, and that Ca2+ and BKCa channels occupy, at constant channel density, a greater fraction of the membrane area in high-frequency cells than in low-frequency cells.

Two components of transducer adaptation in auditory hair cells.

Mechanoelectrical transducer currents in turtle auditory hair cells adapted to maintained stimuli via a Ca(2+)-dependent mechanism characterized by two time constants of approximately 1 and 15 ms. The time course of adaptation slowed as the stimulus intensity was raised because of an increased prominence of the second component. The fast component of adaptation had a similar time constant for both positive and negative displacements and was unaffected by the myosin ATPase inhibitors, vanadate and butanedione monoxime. Adaptation was modeled by a scheme in which Ca(2+) ions, entering through open transducer channels, bind at two intracellular sites to trigger independent processes leading to channel closure. It was assumed that the second site activates a modulator with 10-fold slower kinetics than the first site. The model was implemented by computing Ca(2+) diffusion within a single stereocilium, incorporating intracellular calcium buffers and extrusion via a plasma membrane CaATPase. The theoretical results reproduced several features of the experimental responses, including sensitivity to the concentration of external Ca(2+) and intracellular calcium buffer and a dependence on the onset speed of the stimulus. The model also generated damped oscillatory transducer responses at a frequency dependent on the rate constant for the fast adaptive process. The properties of fast adaptation make it unlikely to be mediated by a myosin motor, and we suggest that it may result from Ca(2+) binding to the transducer channel or a nearby cytoskeletal element.

Electrical response properties of avian lagena type II hair cells: a model system for vestibular filtering.

Data presented represent the first electrical recordings from avian lagena type II hair cells. The perforated-patch variant of the whole cell recording technique was used to investigate how the macroscopic currents shaped the voltage response of the hair cells. Voltage-clamp data separated cells into two broad classes on the basis of differences in activation rates, rates and degree of inactivation, and pharmacological sensitivity. Current-clamp recordings revealed low-quality membrane voltage oscillations (Qc < 1) during pulse current injections. Oscillation frequency correlated with activation rate of the macroscopic currents. The quality of membrane oscillations (Qc) varied linearly with frequency for cells with little inactivation. For cells with rapid inactivation, no relationship was found between Qc and frequency. Rapid inactivation may serve to extend the bandwidth of vestibular hair cells. The frequency measured from voltage responses to pulsed currents may reflect the corner frequency of the cell. The filtering properties of avian lagena hair cells are like those found in all other vestibular end organs, suggesting that the electrical membrane properties of these cells are not responsible for specializing them to a particular stimulus modality.

The effect of early ambulation on hematoma formation and vascular complications following 7 French diagnostic cardiac catheterization.

OBJECTIVE: To assess the feasibility and safety of early ambulation 3 to 4 h after diagnostic 7 French cardiac catheterization. DESIGN: Randomized, single-blind assignment to one of 3, 4 or 6 h ambulation postcardiac catheterization groups. SETTING: Tertiary care community hospital in an urban region. PATIENTS: Eight hundred and seventy-four consecutive inpatients and out-patients presenting for routine diagnostic cardiac catheterization. INTERVENTION: Hematoma formation and other vascular complications recorded at the time of discharge and 24 h later. MAIN RESULTS: No significant difference in hematoma formation rates was noted among patients mobilized at 3 h (3.6%), 4 h (4.8%) or 6 h (3.2%). Late hematoma formation occurred in 2.3% of patients. Other vascular complications were very rare. Reported rates of hematoma formation varied significantly (P < 0.05) among physicians, ranging from 0.9% to 8.0%. CONCLUSIONS: Early ambulation of patients 3 to 4 h after routine diagnostic 7 French cardiac catheterization is both safe and feasible. These findings could result in more efficient recovery bed utilization, reduced nursing costs and improved patient compliance with bed rest.

The endogenous calcium buffer and the time course of transducer adaptation in auditory hair cells.

Mechanoelectrical transducer currents in turtle auditory hair cells adapt to maintained stimuli via a Ca2+-dependent mechanism that is sensitive to the level of internal calcium buffer. We have used the properties of transducer adaptation to compare the effects of exogenous calcium buffers in the patch electrode solution with those of the endogenous buffer assayed with perforated-patch recording. The endogenous buffer of the hair bundle was equivalent to 0.1-0.4 mM BAPTA and, in a majority of cells, supported adaptation in an external Ca2+ concentration of 70 microM similar to that in turtle endolymph. The endogenous buffer had a higher effective concentration, and the adaptation time constant was faster in cells at the high-frequency end than at the low-frequency end of the cochlea. Experiments using buffers with different Ca2+-binding rates or dissociation constants indicated that the speed of adaptation and the resting open probability of the transducer channels could be differentially regulated and imply that the endogenous buffer must be a fast, high-affinity buffer. In some hair cells, the transducer current did not decay exponentially during a sustained stimulus but displayed damped oscillations at a frequency (58-230 Hz) that depended on external Ca2+ concentration. The gradient in adaptation time constant and the tuned transducer current at physiological levels of calcium buffer and external Ca2+ suggest that transducer adaptation may contribute to hair cell frequency selectivity. The results are discussed in terms of feedback regulation of transducer channels mediated by Ca2+ binding at two intracellular sites.

Calcium permeation of the turtle hair cell mechanotransducer channel and its relation to the composition of endolymph.

1. Recordings of mechanoelectrical transducer currents were combined with calcium imaging of hair bundles in turtle auditory hair cells located near the high-frequency end of the cochlea. The external face of the hair bundles was perfused with a range of Ca2+ concentrations to study the quantitative relationship between Ca2+ influx and transducer adaptation. 2. With Na+ as the monovalent ion, the peak amplitude of the transducer current decreased monotonically as the external [Ca2+] was raised from 25 microns to 20 mm. When Na+ was replaced with the impermeant Tris the transducer current increased with external [Ca2+]. These results indicate that Ca2+ can both permeate and block the transducer channels. The Ca2+ concentration for half-block of the monovalent current was 1 mm. 3. To quantify the Ca2+ influx, the fraction of transducer current carried by Ca2+ was measured using the change in bundle fluorescence in cells loaded with 1 mm Calcium Green-1. The fluorescence change was calibrated by substituting an impermeable monovalent ion to render Ca2+ the sole charge carrier. 4. In the presence of Na+, the fractional Ca2+ current was approximately 10% in 50 microns Ca2+, a concentration similar to that in endolymph, which bathes the hair bundles in vivo. The amount of Ca2+ entering was dependent on the identity of the monovalent ion, and was larger with K+, suggesting that the transducer channel is a multi-ion pore. 5. Over a range of ionic conditions, the rate of transducer adaptation was proportional to Ca2+ influx indicating that adaptation is driven by a rise in intracellular [Ca2+]. 6. Shifts in the current-displacement function along the displacement axis in different external Ca2+ concentrations were predictable from variation in the resting Ca2+ influx. We suggest that changes in the resting open probability of the transducer channels adjust the entry of Ca2+ to keep its concentration constant at an internal site. 7. The results demonstrate that endolymph containing high K+, 50 microns Ca2+ and low Mg2+ concentrations, maximizes the transducer current while still allowing sufficient Ca2+ entry to drive adaptation. The hair cell mechanotransducer channel, in its permeation and block by Ca2+, shows behaviour similar to the voltage-gated Ca2+ channel and the cyclic nucleotide-gated channel.