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.

Decoupled hair bundles of the bullfrog (Lithobates catesbeianus) sacculus exhibit spontaneous oscillations in vitro. We examine the effect of the somatic electrical circuit upon active hair bundle motility. We found that innate bundle movements exhibit a complex profile with multiple periodicities. Inhibition of somatic ion channels using targeted neurotoxins and modified physiological solutions strongly affects the bundles' mechanical behavior, modifying the amplitude and the temporal characteristics of the oscillation profile.

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.

A Complementary Metal Oxide Semiconductor (CMOS) camera (1024x1024 pixels) is used to record spontaneous oscillations of hair cell stereocillia in an in-vitro preparation of the bullfrog sacculus with the otolithic membrane removed. The CMOS camera is attached to an Olympus BX51WI Microscope inside of a sound-isolation chamber, with white light transmission illumination using an X-Cite 120 metal halogenide lamp. The combination of the parallel readout of the CMOS chip and the high intensity of illumination allows full frame images of the oscillations to be taken at 1000 frames per second. A weighted, time averaged differential algorithm is used to aid in the visualization of the hair cell movement. To detect the displacement from its center of the stereocillia tip with nanometer position resolution and millisecond time resolution, an average background intensity value was subtracted from each image to remove lamp intensity fluctuations and then a center of intensity algorithm was applied. This combination of our imaging system and data analysis allows for the oscillations of more than one hair cell to be recorded during the same time period, and their frequency components extracted.