Dolores Bozovic

Magnetic nanoparticles for ultrafast mechanical control of inner ear hair cells.

We introduce cubic magnetic nanoparticles as an effective tool for precise and ultrafast control of mechanosensitive cells. The temporal resolution of our system is ∼1000 times faster than previously used magnetic switches and is comparable to the current state-of-the-art optogenetic tools. The use of a magnetism-gated switch reported here can address the key challenges of studying mechanotransduction in biological systems. The cube-shaped magnetic nanoparticles are designed to bind to components of cellular membranes and can be controlled with an electromagnet to exert pico-Newtons of mechanical force on the cells. The cubic nanoparticles can thus be used for noncontact mechanical control of the position of the stereocilia of an inner ear hair cell, yielding displacements of tens of nanometers, with sub-millisecond temporal resolution. We also prove that such mechanical stimulus leads to the influx of ions into the hair cell. Our study demonstrates that a magnetic switch can yield ultrafast temporal resolution, and has capabilities for remote manipulation and biological specificity, and that such magnetic system can be used for the study of mechanotransduction processes of a wide range of sensory systems.

Distribution of Frequencies of Spontaneous Oscillations in Hair Cells of the Bullfrog Sacculus

Under in vitro conditions, free-standing hair bundles of the bullfrog (Rana catesbeiana) sacculus have exhibited spontaneous oscillations. We used a high-speed complementary metal oxide semiconductor camera to track the active movements of multiple hair cells in a single field of view. Our techniques enabled us to probe for correlations between pairs of cells, and to acquire records on over 100 actively oscillating bundles per epithelium. We measured the statistical distribution of oscillation periods of cells from different areas within the sacculus, and on different epithelia. Spontaneous oscillations exhibited a peak period of 33 ms (+29 ms, -14 ms) and uniform spatial distribution across the sacculus.

Multiple-Timescale Dynamics Underlying Spontaneous Oscillations of Saccular Hair Bundles

Spontaneous oscillations displayed by hair bundles of the bullfrog sacculus have complex temporal profiles, not fully captured by single limit-cycle descriptions. Quiescent intervals are typically interspersed with oscillations, leading to a bursting-type behavior. Temporal characteristics of the oscillation are strongly affected by imposing a mechanical load or by the application of a steady-state deflection to the resting position of the bundle. Separate spectral components of the spontaneous motility are differently affected by increases in the external calcium concentration. We use numerical modeling to explore the effects of internal parameters on the oscillatory profiles, and to reproduce the experimental modulation induced by mechanical or ionic manipulation.

Dynamic state and evoked motility in coupled hair bundles of the bullfrog sacculus.

Spontaneous oscillations, one of the signatures of the active process in non-mammalian hair cells, have been shown to occur in individual hair bundles that have been fully decoupled from the overlying membrane. Here we use semi-intact preparations of the bullfrog sacculus to demonstrate that under more natural loading conditions, innate oscillations are suppressed by the presence of the overlying otolithic membrane, indicating that hair bundles lie in the quiescent rather than the unstable regime. Transepithelial electrical stimulation was then used to test the effect of evoking entrained hair bundle movement with an external stimulus. Firstly, we used a preparation in which the otolithic membrane has been partially detached, coupling only hair bundles of comparable orientations. Secondly, we deposited artificial polymer membranes on top of the epithelium so as to connect to only 10-20 cells. In both of these systems, hair bundle motion phase-locked by the electrical signal was found to induce movement in the overlying structures.

Correlated movement of hair bundles coupled to the otolithic membrane in the bullfrog sacculus.

High-speed imaging with a CMOS camera was used to track the motion of multiple hair bundles of the bullfrog sacculus. To maintain the natural degree of intercell coupling, the overlying otolithic membrane was left intact atop the in vitro preparation. Effects of an incoming mechanical signal were mimicked by laterally deflecting the membrane with a glass probe at physiological amplitudes. The motion evoked in the underlying hair bundles was found to be highly phase-locked, yielding an entrained response across hundreds of cells. We imaged significant portions of the saccular epithelium, up to 40 x 350 microm(2), and observed a high degree of correlation over those scales.

Coupling and elastic loading affect the active response by the inner ear hair cell bundles.

Active hair bundle motility has been proposed to underlie the amplification mechanism in the auditory endorgans of non-mammals and in the vestibular systems of all vertebrates, and to constitute a crucial component of cochlear amplification in mammals. We used semi-intact in vitro preparations of the bullfrog sacculus to study the effects of elastic mechanical loading on both natively coupled and freely oscillating hair bundles. For the latter, we attached glass fibers of different stiffness to the stereocilia and observed the induced changes in the spontaneous bundle movement. When driven with sinusoidal deflections, hair bundles displayed phase-locked response indicative of an Arnold Tongue, with the frequency selectivity highest at low amplitudes and decreasing under stronger stimulation. A striking broadening of the mode-locked response was seen with increasing stiffness of the load, until approximate impedance matching, where the phase-locked response remained flat over the physiological range of frequencies. When the otolithic membrane was left intact atop the preparation, the natural loading of the bundles likewise decreased their frequency selectivity with respect to that observed in freely oscillating bundles. To probe for signatures of the active process under natural loading and coupling conditions, we applied transient mechanical stimuli to the otolithic membrane. Following the pulses, the underlying bundles displayed active movement in the opposite direction, analogous to the twitches observed in individual cells. Tracking features in the otolithic membrane indicated that it moved in phase with the bundles. Hence, synchronous active motility evoked in the system of coupled hair bundles by external input is sufficient to displace large overlying structures.

Phase-locked spiking of inner ear hair cells and the driven noisy Adler equation.

The inner ear constitutes a remarkably sensitive mechanical detector. This detection occurs in a noisy and highly viscous environment, as the sensory cells—the hair cells—are immersed in a fluid-filled compartment and operate at room or higher temperatures. We model the active motility of hair cell bundles of the vestibular system with the Adler equation, which describes the phase degree of freedom of bundle motion. We explore both analytically and numerically the response of the system to external signals, in the presence of white noise. The theoretical model predicts that hair bundles poised in the quiescent regime can exhibit sporadic spikes—sudden excursions in the position of the bundle. In this spiking regime, the system exhibits stochastic resonance, with the spiking rate peaking at an optimal level of noise. Upon the application of a very weak signal, the spikes occur at a preferential phase of the stimulus cycle. We compare the theoretical predictions of our model to experimental measurements obtained in vitro from individual hair cells. Finally, we show that an array of uncoupled hair cells could provide a sensitive detector that encodes the frequency of the applied signal.

Voltage-Mediated Control of Spontaneous Bundle Oscillations in Saccular Hair Cells.

Hair cells of the vertebrate vestibular and auditory systems convert mechanical inputs into electrical signals that are relayed to the brain. This transduction involves mechanically gated ion channels that open following the deflection of mechanoreceptive hair bundles that reside on top of these cells. The mechano-electrical transduction includes one or more active feedback mechanisms to keep the mechanically gated ion channels in their most sensitive operating range. Coupling between the gating of the mechanosensitive ion channels and this adaptation mechanism leads to the occurrence of spontaneous limit-cycle oscillations, which indeed have been observed in vitro in hair cells from the frog sacculus and the turtle basilar papilla. We obtained simultaneous optical and electrophysiological recordings from bullfrog saccular hair cells with such spontaneously oscillating hair bundles. The spontaneous bundle oscillations allowed us to characterize several properties of mechano-electrical transduction without artificial loading the hair bundle with a mechanical stimulus probe. We show that the membrane potential of the hair cell can modulate or fully suppress innate oscillations, thus controlling the dynamic state of the bundle. We further demonstrate that this control is exerted by affecting the internal calcium concentration, which sets the resting open probability of the mechanosensitive channels. The auditory and vestibular systems could use the membrane potential of hair cells, possibly controlled via efferent innervation, to tune the dynamic states of the cells. SIGNIFICANCE STATEMENT The sensation of sound and balance starts by converting minute mechanical motions into electrical signals. This is accomplished by sensory hair cells, in which the opening and closing of mechanosensitive channels follows the motion of their hair bundles. These hair bundles can exhibit motility without an external drive. Underlying these spontaneous bundle oscillations are two coupled feedback mechanisms that improve the cells response when mechanically stimulated. Here, we present synchronized optical and electrical recordings from cells with spontaneously oscillating bundles and show that the membrane potential may be a control parameter, tuning the cells feedback processes. This control comes from modulation of the channels open probability, which is affected by the concentration of calcium inside the cell.

Low Frequency Entrainment of Oscillatory Bursts in Hair Cells

Sensitivity of mechanical detection by the inner ear is dependent upon a highly nonlinear response to the applied stimulus. Here we show that a system of differential equations that support a subcritical Hopf bifurcation, with a feedback mechanism that tunes an internal control parameter, captures a wide range of experimental results. The proposed model reproduces the regime in which spontaneous hair bundle oscillations are bistable, with sporadic transitions between the oscillatory and the quiescent state. Furthermore, it is shown, both experimentally and theoretically, that the application of a high-amplitude stimulus to the bistable system can temporarily render it quiescent before recovery of the limit cycle oscillations. Finally, we demonstrate that the application of low-amplitude stimuli can entrain bundle motility either by mode-locking to the spontaneous oscillation or by mode-locking the transition between the quiescent and oscillatory states.

Mechanical Overstimulation of Hair Bundles: Suppression and Recovery of Active Motility

We explore the effects of high-amplitude mechanical stimuli on hair bundles of the bullfrog sacculus. Under in vitro conditions, these bundles exhibit spontaneous limit cycle oscillations. Prolonged deflection exerted two effects. First, it induced an offset in the position of the bundle. Recovery to the original position displayed two distinct time scales, suggesting the existence of two adaptive mechanisms. Second, the stimulus suppressed spontaneous oscillations, indicating a change in the hair bundle’s dynamic state. After cessation of the stimulus, active bundle motility recovered with time. Both effects were dependent on the duration of the imposed stimulus. External calcium concentration also affected the recovery to the oscillatory state. Our results indicate that both offset in the bundle position and calcium concentration control the dynamic state of the bundle.