Projects

Fast adaptation is a process that modulates the hair cell dynamic range with kinetics measured down to the 10s of microseconds. These rapid kinetics limit potential mechanisms. Although multiple theories exist as to the mechanism of fast adaptation, we are no closer to explicitly understanding either its function or the molecular underpinnings responsible for its manifestation.

The lipid bilayer has intrinsic mechanical properties based on which lipid components are present as well as the underlying interactions of lipids with membrane and cytoskeletal proteins. Alterations in lipids can affect hair cell mechanotransduction either directly by interacting with the proteins involved in the process or indirectly through the intrinsic mechanical properties of the bilayer. We are investigating lipid properties in multiple ways that include:

a) Pharmacologically removing or adding specific lipids to the stereocilia while measuring MET currents or lipid diffusivity.

b) Altering lipid-cytoskeletal interactions again while monitoring lipid diffusivity and MET currents.

c) Identifying specific lipid components of the stereocilia and their localization within these stereocilia.

In situ, outer hair cell bundles are embedded into a tectorial membrane while inner hair cell bundles remain free standing, though in a confined space under the tectorial membrane. Movement of the basilar membrane exerts force onto the hair bundles that to date is considered a sheering force; however, recent data questions this long-standing theorem. Mammalian cochlear hair bundles show a lack of coherence, that allow them to splay when stimulated. In addition, the shape of the hair bundles suggest that shearing would not provide stimulation in the most sensitive stereocilia direction. We are using high speed imaging (up to 100k Hz) coupled with high speed stimulation of the cochlea in situ to directly measure hair bundle motion.

Multiple functions have been ascribed to the synaptic ribbon. These functions include: maintaining a high number of vesicles near release sites, being a site for generation of new synaptic vesicles, controlling multivesicular release, regulating timing of vesicle fusion and creating a barrier to calcium diffusion. We are taking a multidisciplinary approach to unravelling the underlying functions and associated molecular mechanisms. We use a multisine stimulus paradigm to track vesicle fusion by monitoring membrane capacitance. We use postsynaptic recordings to assess synaptic function. We are also using molecular tools such as voltage sensors to track activity in multiple fibers, and genetic tools such as ribeye knockout animals to investigate the function of the synaptic ribbon. High speed confocal calcium imaging coupled with peptides that label synapses are also included in these studies. And finally, we include immunocytochemistry and electron microscopy.

Aminoglycoside is antibiotics used to treat gram-negative bacterial infections. This drug saves people from life-threatening infection. However, it has side effects which are ototoxicity and nephrotoxicity. Even though nephrotoxicity is reversible, ototoxicity is permanent. The ototoxicity occurs in as many as 20% of patients who received aminoglycoside intravenously for multiple days. Our goal of this study is to determine aminoglycoside trafficking route into cochlea by in vivo monitoring of aminoglycoside and investigate a blocking strategy of it.