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

Control of hearing sensitivity by tectorial membrane calcium

When sound stimulates the stereocilia on the sensory cells in the hearing organ, Ca2+ ions flow through mechanically gated ion channels. This Ca2+ influx is thought to be important for ensuring that the mechanically gated channels operate within their most sensitive response region, setting the fraction of channels open at rest, and possibly for the continued maintenance of stereocilia. Since the extracellular Ca2+ concentration will affect the amount of Ca2+ entering during stimulation, it is important to determine the level of the ion close to the sensory cells. Using fluorescence imaging and fluorescence correlation spectroscopy, we measured the Ca2+ concentration near guinea pig stereocilia in situ. Surprisingly, we found that an acellular accessory structure close to the stereocilia, the tectorial membrane, had much higher Ca2+ than the surrounding fluid. Loud sounds depleted Ca2+ from the tectorial membrane, and Ca2+ manipulations had large effects on hair cell function. Hence, the tectorial membrane contributes to control of hearing sensitivity by influencing the ionic environment around the stereocilia.Funding Agencies|Swedish Research Council [2013-03403, 2017-06092]; Torsten Soderberg Foundation; Tysta Skolan Foundation; AFA Forsakrings AB; County Council of Ostergotland; US National Institute on Deafness and Other Communication Disorders [DC 00141]; Wenner-Gren Foundation</p

Experimental setup.

<p>(A) Top-down view of a freestanding hair bundle with a glass probe (black line positioned vertically across the image) attached to the tallest row of stereocillia. Because of light piping, the stereocillia appear much brighter than the background. Scale bar: 2.5 m. (B) Schematic diagram showing a side view of a hair bundle with a probe attached. The piezoelectric actuator displaces the probe's base in the direction of the bundle's axis of sensitivity indicated by the arrow in the figure. (C) Top-down view of hair bundles coupled to the otolithic membrane. The pits in the membrane into which the bundles protrude give rise to the bright ellipses around each bundle. The shadow of the probe's tip has been highlighted with the dashed line. Scale bar: 5 m. (D) Schematic diagram of a side view of hair bundles coupled to the otolithic membrane and stimulated with a probe. In the actual experiment, the probe's tip is embedded a few microns into the otolithic membrane. Note than in C and D, the probes have not been drawn to scale; in particular, the cantilever arms are typically a few hundred microns in length.</p

Frequency selectivity of the phase-locked response under mechanical loading.

<p>A discrete frequency sweep was applied to each bundle with the attached glass probe. Each sweep ranged from 5–50 Hz in 1 Hz increments, with 5 cycles presented at each frequency. The frequency trains were applied at increasing amplitudes. The phase-locked response was extracted for each frequency and amplitude and displayed in a color-coded plot, with the range shown to the right of each panel. Under light loading (100–200 N/m), the bundles exhibited tuning around their characteristic frequencies. Under stiffer loading (400 N/m and higher), the bundles displayed no tuning. Note that the plots contain only 1∶1 mode-locking; higher-order modes were not included. Traces were recorded at 500 fps.</p

The magnitude of the twitch scales linearly with phase-locked amplitude.

<p>(A) and (B) Scatter plots of the active movement or twitch versus the phase-locked amplitude for positive and negative deflections. Data have been pooled from five preparations. All recordings were taken at 5000 fps.</p

Coupling of active hair bundles by the otolithic membrane broadens the response to sinusoidal stimulation.

<p>(A) A top-down view of a 15050 m area of the saccular epithelium with the otolithic membrane removed. The linear response function averaged over the four bundles in the corners of the white square is shown in panel C (the central bundle was partially occluded by an otolith). The tallest row of stereocilia appear as elongated bright features. The kinociliary bulbs (indicated by arrows) appear as grey circles to the right of the stereocilia. Scale bar: 10 m. (B) Normalized linear response functions for three hair bundles coupled to the otolithic membrane. The mean and standard deviation at each frequency were computed from a digitally resampled trace on a cycle-by-cycle basis over the 1 s recording. When the otolithic membrane was removed from this sample, 84% of bundles exhibited spontaneous oscillations demonstrating that the active process was maintained throughout the measurement. C. Ensemble-averaged ( = 4) linear response function of bundles coupled to the otolithic membrane from the preparation shown in panel A. Filled squares: the average response was essentially flat across the physiological range of frequencies. Open circles: Blocking the transduction channels with 20 M gentamicin had little effect on the response at low frequencies; the response was modestly reduced at frequencies above 150 Hz. With the otolithic membrane removed, 90% of the bundles in this preparation oscillated spontaneously. All recordings were taken at 1000 fps.</p

Active hair bundle movement under the otolithic membrane.

<p>(A)Traces of twitches in underlying hair bundles evoked by a series of 5 ms pulses delivered to the otolithic membrane in the excitatory (top) and inhibitory (bottom) directions. The command signals were half-cycles of a 100 Hz sine wave and are shown below the traces. (B) With the transduction channels blocked with 20 M gentamicin, the active movement was abolished. The probe had a stiffness of  = 1200 N/m. (C) and (D) Average traces for the bundle movement shown in panels A and B. (E) The averaged traces obtained from 1 m aluminum oxide beads dispersed on the otolithic membrane of the same preparation. (F) Average traces of a pit boundary measured in a different preparation. In both E and F, the otolithic membrane was entrained by the active bundle movement at the end of the applied pulse. The stimulus fiber used on the sample in panel F had a stiffness of  = 1400 N/m. In C, E, and F the exponential fits (red lines) from which the recovery times were extracted, have been overlaid on the traces. (G) Response of a hair bundle which showed a 15 nm twitch in response to sinusoidal pulses. (H) +10 A transepithelial electric current had little effect on either phase-locked amplitude or the twitch for both stimuli. (I) −10 A current reduced the magnitude of the twitch to 10 nm for positive deflections but had a negligible effect for negative displacements. In G, H, and I the average traces have been plotted below the raw data.</p