Determining how the natural hair bundle stimulations shapes the hair cell receptor current - Abstract: Auditory and vestibular sensory cells use the hair bundle, a stair-cased array of actin-filled stereocilia, to translate hair bundle motion into an electrical signal. Mechanically gated (MET) ion channels are activated by force created by the pulling of a tip link that extends between stereocilia. Coordinated stimulation of tip links regulates multiple channel openings to generate a receptor current. Mammalian cochlear hair bundles unexpectedly were found to have weak interstereocilia connections which sensitize them to the stimulus mode. The cochlea machine is designed to stimulate hair bundles in a manner that maximizes their sensitivity and frequency selectivity, understanding how hair bundles are stimulated is critical to our understanding of cochlear mechanics. We seek to understand how the mechano-electrical and electromechanical feedback between the hair bundle, its environment and outer hair cell electromotility regulate sensitivity and selectivity of the cochlear output. A first step in unraveling these interactions is to understand and document how hair bundles move in situ and how this motion shapes the receptor current. This proposal bridges from the system level cochlear mechanics to how hair bundle motion shapes the receptor current. We are uniquely positioned to investigate these questions because we have developed the technology and created collaborations to image hair bundles (even stereocilia in inner hair cells), tectorial membrane (TM) and reticular lamina (RL), in situ and in vitro. We are coupling the high spatial resolution of imaging XY with optical coherence tomography which has high vibrational resolution in Z but poor spatial resolution and with low coherence interferometry that has better spatial resolution and excellent vibrational sensitivity. Together these tools provide a powerful platform in which to validate our in vitro preparation and to investigate cochlea vibrational patterns, providing the first measurements of mature hair bundle motion in situ. We will leverage this data to investigate MET receptor currents in vitro to directly assess hair bundle filtering properties. This proposal will address three important topics: How are hair bundles stimulated in situ (SA1)? Which hair bundle components most impact in situ motion (SA2)? How are receptor currents modified by natural hair bundle stimulation (SA3). Specific Aim 1 measures motion in situ from mice aged P10-15 and P24-35. These data, assisted by physics-based models of motion will determine the mode of hair bundle stimulation and how hair bundle motion relates to TM and RL motion. SA2 probes a set of genetically altered mice, to identify the hair bundle and TM components driving the hair bundle response. SA3 develops stimulus technology to mimic in situ stereocilia motion for IHC and OHC bundles in vitro to assess receptor current properties under physiological conditions using physiologically relevant modes of stimulation. These data provide insight into cochlear mechanics and into how hair bundles are specialized to best respond to their mode of stimulation. The hair bundle is the site of many genetic disorders and the target of noise and age-related hearing loss. This proposal provides foundational data needed for developing treatment strategies.
