Fast Adaptation

Now that we know that fast adaptation is not dependent on calcium entry, what is the mechanism of this process and what is its physiological relevance?

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.



PIP2 is a lipid known to interact with many ion channels including a range of mechanically sensitive ion channels. Does PIP2 directly interact with hair cell mechanotransduction machinery?

PIP2 is enriched at the tops of stereocilia, right near to the site of mechanotransduction. We know that PIP2 can alter MET channel properties such as single channel conductance and ion permeation. We also know that it is required for fast adaptation. We are presently trying to identify the interacting molecules of PIP2 to unravel toe specific molecular mechanisms of these processes.


Lipid Bilayer

What role does the lipid bilayer play in modulating mechanotransduction response properties? Do bilayer mechanical properties alter force transfer to the mechanotransduction channel? Are there specific protein lipid interactions that underlie this force transfer?

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.


Hair Bundle Properties

What are the intrinsic properties of hair bundles that dictate their output? How does hair bundle cohesiveness shape the hair cell receptor potential?

Intrinsic hair bundles shape force transfer to the mechanotransduction channel such that the type of stimulus used results in a very different output. The consequences of these output are critical in that they directly shape the hair cell receptor potential. We are characterizing the mechanical properties of both IHC and OHC hair bundles to identify the most relevant mechanical properties as well as the molecular machinery creating these properties. To address these questions, we are using specially designed piezoelectric devices and fluid jet systems for mechanically stimulating the hair bundle why measuring hair bundle movements with high speed cameras or high speed confocal images that monitor calcium entry through the mechanically gated channels. We simultaneously monitor the MET currents.


Hair Bundle Movement

How does a hair bundle move when stimulated in situ? How does the tectorial membrane shape this motion?

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.


Vesicle Fusion

How does the synapse maintain high rates of vesicle fusion?

By developing new technology to monitor vesicle fusion in real time, we identified a component of release that requires vesicle trafficking and are now identifying the molecular mechanisms responsible for this release component. Here to we use electrophysiological, pharmacological, genetic and optical tools to delineate mechanisms underlying synaptic vesicle trafficking.


Synaptic Ribbons

Hair cells contain an osmophilic dense body, often called a synaptic ribbon, to which many functions have been ascribed.
We are directly investigating the function of the dense body using whole animal and single cell electrophysiology both pre and post synaptic recordings, super resolution quantitative immunocytochemistry, high speed confocal calcium imaging and genetic manipulations.

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.


Afferent Fiber Properties

What underlies the broad range of afferent fiber properties?
What are the pre-and post synaptic specializations that underlie the diversity in these properties?

Afferent fibers have different sensitivities to sound input. We are determining whether these differences occur pre or post synaptically and whether these fibers are divided morphologically around the hair cell.


Imaging of aminoglycoside uptake

How do aminoglycosides move into the cochlea? How do we block ototoxicity induced by aminoglycosides?

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.


Calcium (Ca2+) imaging

How many hair cells are activated with a pure tone? How do neighbor hair cells act? What changes does loud sound make?

Sound induces Ca2+ influx into inner hair cells through L-type voltage-gated Ca2+ channels, leading to the release of neurotransmitter. To study sound-evoked Ca2+ responses in the cochlea, we use the GECI GCaMP6 expressed selectively in hair cells. Our goal of this study is to determine dynamics of peripheral auditory encoding by functional monitoring of multiple cochlear cells