Damien Ramunno-Johnson

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.

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.

Magnetic Bead Actuation of Saccular Hair Cells

When decoupled from the overlying membrane, hair bundles of the amphibian sacculus exhibit spontaneous oscillation. To explore the dynamics of this innate motility without an imposed external load, we recorded their oscillations with a high‐speed CMOS camera, and applied mechanical manipulation that minimally alters the geometry of an individual hair bundle. We present a technique that utilizes micron‐sized magnetic particles to actuate the stereociliary bundle with a magnetized probe. Quasi‐steady‐state displacements were imposed on freely oscillating bundles. Our data indicate that deflection of the bundle affects both the frequency and the amplitude of the oscillations, with a crossing of the bifurcation that is dependent on the direction and speed of the applied offset.

Exploring the Electrical Resonance's Affect on the Mechanical Oscillations of Hair Cells in the Bullfrog Sacculus

Under in vitro conditions, uncoupled hair bundles of the bullfrog (Rana catesbeiana) sacculus have been shown to exhibit spontaneous oscillations. We used a high-speed complementary metal oxide semiconductor camera to track the movements of hundreds of cells in parallel from dozens of preparations. This work revealed that the spontaneous oscillations exhibit multiple timescales with a slow modulation on a rapid oscillation. Experiments inhibiting the electrical resonance in the cell body show a strong effect on the mechanical oscillations of the hair bundles. This indicates that the electrical oscillation is coupled with the mechanical oscillations of the hair bundles.