William E. Brownell - US grants

Neural communication among rhombencephalic auditory structures is being explored by means of single-unit recordings in the decerebrate, unanesthetized cat. The response characterisitcs of central nervous system axons that are efferent from and afferent to the cochlear nuclei are examined. Both extracellular and intracellular recordings are made. A few fibers are injected with horseradish peroxidase after physiological characterization and their somatic origin and terminal distributions determined. Fiber response characteristics are compared with single unit responses previously recorded in our laboratory from the cochlear nuclei and superior olivary complex. The studies examine neuronal circuits involved in the formation of receptive field features of the principal cells of the dorsal cochlear nucleus, octopus cells of the posteroventral cochlear nucleus, and neurons of the superior oblivary complex. The experiments test for the possibility that axonal response characteristics differ from those recorded somatically. The neural substrates, spike train coding and synaptic mechanisms by which information is abstracted in the central auditory system (including mechanisms for binaural interactions) may be revealed.

The long range goal of this project is to determine the role(s) of outer hair cells in mammalian hearing. Their contribution to cochlear transduction is analyzed by measuring changes in inner ear currents resulting from biochemical and pharmacological manipulations that are known to affect outer hair cells. The biophysics of these changes is investigated directly using isolated cell culture preparations. The major energy source for the sensory and motor functions of the outer hair cell comes from the "silent current" generated by- metabolically active ion pumps in the stria vascularis. The silent current is modulated by conductance changes in outer hair cell membranes. Parallel in vivo and in vitro experiments measure the electrical and chemical gradients at the organ and cellular level respectively. Electrical potentials are recorded in vivo at fixed intervals along a microelectrode track and the current density is calculated by taking the first spatial derivative of the potential field. The resulting analysis permits a complete three dimensional characterization of the strial current across the cochlear partition in silence and during acoustic stimulation. The effect of medial olivocochlear component efferent fiber stimulation on the standing current is measured and can be attributed to a direct action on outer hair cells. Perfusion of the cochlea with putative efferent neurotransmitter substances may provide additional insight into the postulated sensory-motor feedback loop that adjusts the electro-mechanical pin of the organ of Corti. In vitro measurements are made of the effect of putative efferent neurotransmitters on the outer hair cell's unique hydraulic support system. Their effects on rapid electromotility and on whole cell conductivity are also measured using a variety of modern recording techniques. These include whole cell recording, and the analysis of images obtained using either video enhanced microscopy or a laser scanning confocal microscope. Our results are described and extended by developing models of the outer hair cell's static and dynamic mechanics. The models, in turn. suggest critical experiments to verify hypotheses. Aspirin ototoxicity and other I manipulations affect rapid electromotility by changing the cell's turgor pressure. The mechanisms of outer hair cell volume regulation are investigated by measuring the effect of aspirin. aminoglycosides and other ototoxic drugs as well as simple sugars on cell volume and turgor pressure. Clarification of their action on the isolated cell coupled with recordings made while perfusing the cochlea with these agents in vivo will lead to a better understanding of the pathology of the ototoxic drugs. Our measurements of extracellular, transmembrane and intracellular potential and chemical gradients in the cochlea will clarify the sensory and motor contributions of the outer hair cell in cochlear transduction and help to explain the basis for hearing loss associated with outer hair cell pathology.

DESCRIPTION (provided by applicant): The long-term goal of this research is to study the physical determinants of outer hair cell electromotility. The outer hair cell enhances the sensitivity and frequency selectivity of mammalian hearing by converting the energy of intracochlear electrochemical gradients into mechanical energy. The motor mechanism responsible resides in the outer hair cell's lateral wall, a 100 nanometer thick, three-layer structure composed of two membranes with a cytoskeletal network sandwiched between them. Energy conversion by the lateral wall is bidirectional (electrical to mechanical and mechanical to electrical). The tight coupling between electrical polarization and mechanical displacement is characteristic of piezoelectricity. The specific objectives of this project period are to 1) identify the mechanisms that contribute to the high frequency membrane potentials that drive electromotility and 2) to identify the contributions of the lateral wall to the modulation and maintenance of the electrochemical gradients necessary for cell function. Coordinated theoretical and experimental approaches identify the piezoelectric and ionic contributions to the cell's high frequency response. They also address how the unique molecular organization of the lateral wall influence the diffusion of membrane constituents and contribute to the transport of ions, water and other molecules through the narrow space between its membranes. Experiments will assess the role of the membrane protein prestin by recording from outer hair cells isolated from normal and prestin null mutant mice. The lateral mobility of fluorescent lipid analogues and membrane proteins will be determined. Methods include the development of new transgenic mice, micro electro impedance spectroscopy, voltage and current-clamp, including two-pipette recording; fluorescence recovery after photobleaching, confocal microscopy, total internal reflection photolysis, video microscopy and computational modeling. The studies will further clarify the role of the outer hair cell as the cochlear amplifier. Clarification of the physical principles underlying electromotility will also contribute to the emerging field of biological nanotechnology.

Outer hair cells (OHC) are required for normal mammalian hearing. They amplify sound vibrations in the inner ear through their ability to convert electrical to mechanical energy. Interfering with the force generating mechanism results in hearing loss. The mechanism responsible for this force production is unknown but it resides in the OHC's lateral wall. The lateral wall is an elegant, three layered, composite nanostructure that is as structurally conspicuous in the OHC as the force production. The long-term goal of this proposal is to understand the mechanism by which the nanoscale mechanical anatomy of the lateral wall facilitates OHC electomechanical force production, particularly at acoustic frequencies. Experimental and theoretical approaches will determine how the organization of lateral wall directs mechanical force along the OHC's axis and compensates for viscous damping. We will investigate the characteristics of two plausible molecular mechanisms, one driven by in-plane conformational changes of a motor protein and the other by out-of-plane flexoelectric bending. Contemporary electrophysiological and advanced optical techniques will be used. Specific Aim 1 will test the hypothesis that the elastic properties of the lateral wall vary with membrane potential. Local cell wall strains will be measured with optical tweezers and under conditions of micropipet aspiration and osmotic challenge at different holding potentials. Specific aim 2 will establish the magnitude of out-of-plane bending of the plasma membrane in response to active and passive length changes of the OHC using polarized evanescent illumination. Specific aim 3 is to establish the role of the lateral wall cytoskeleton in maintaining cell shape and transmitting active forces. The cell's electromotile response during damage and recovery of the cytoskeleton will allow a determination of its active force transmission. Specific aim 4 will result in a complete nanoscale model of OHC active force production. It will include full dynamic description of the trilaminar lateral wall mechanical anatomy. The model will allow us to predict force-deformation relationships for the OHC at acoustic frequencies and it will meet all the criteria for the mechanism of OHC force production.

DESCRIPTION (provided by applicant): A research training program in Otolaryngology-Head and Neck Surgery is designed to prepare residents and medical students at the Texas Medical Center in Houston for a career as academic Otolaryngologists and/or researchers. The training faculty includes nine scientists at the Baylor College of Medicine and seven at the MD Anderson Cancer Center. The Baylor College of Medicine faculty consists of 5 members of the Bobby R. Alford Department of Otorhinolaryngology and Communicative Sciences and 4 faculty members from other departments. All 9 Baylor College of Medicine faculty have active research programs in hearing and balance, ranging from molecular biophysics to vestibular rehabilitation. The MD Anderson Cancer Center faculty consists of 3 members of the Department of Head and Neck Surgery and 4 members in other departments; all 7 laboratories are involved in basic and/or translational investigations of neoplastic diseases. Oversight of the training program is provided by an advisory committee consisting of the 2 department chairs and the otolaryngology residency director at the Baylor College of Medicine. The training program has 2 tracks. The first is for otolaryngology residents. One (1) resident in each incoming class will receive 2 contiguous years of research training as part of an integrated 7-year experience that will prepare him/her to balance both clinical and research activities. The second track is for medical students interested in otolaryngology-head and neck surgery and offers either a short-term (2-month) or a 1-year research opportunity in any of the laboratories in the training program. In addition to their laboratory experiences, the training includes coursework relevant to the specific field of investigation, research ethics, and responsible conduct of research on human subjects, as well as grantsmanship; all of which are designed to ensure the production of outstanding researchers.

Outer hair cells (OHC) are required for normal mammalian hearing. They convert electrical to mechanical energy and contribute to the cochlear amplifier that magnifies and refines sound vibrations in the inner ear. Interfering with their electromechanical force generating mechanism results in hearing loss. The mechanism responsible for this force production resides in the OHC's plasma membrane. All membranes appear able to convert electrical into mechanical force. The OHC membrane contains a protein called prestin thatenhances the process. In addition, the cylindrical OHCs are effective at taking the membrane generated force and directing it parallel to their longitudinal axis. This is achieved by an elegant, three layered, composite nanostructure that is as biologically unique as the role it plays in hearing. One goal of this proposal is to understand how prestin and the nanoscale mechanical anatomy of the lateral wall facilitate OHC electromechanical force production, particularly at acoustic frequencies. The role of prestin is determined by examining how its presence or absence alters electromechanical transduction. The effect of drugs and changes in the intracellular chloride concentration known to alter electromechanical transduction in the OHC and thereby modulate hearing are assessed for all experimental manipulations. Coordinated experimental, mathematical and computational approaches will determine how the organization of the lateral wall directs the electromechanical force generated by the plasma membrane. Three specific aims investigate OHC mechanical properties and electromechancial transduction at progressively larger scales beginning with the cell membrane and ending with the whole cell. Voltage-clamp and optical tweezers are used together in all three specific aims. Specific Aim 1 examines electromechanical transduction in the plasma membrane by investigating the mechanical response of membrane tethers to changes in the transmembrane potential. These experiments will determine the electromechanical transduction coefficient for the membrane alone. Specific Aim 2 probes the response of the lateral wall and Specific Aim 3 examines the mechanics and electromechanics of the whole cell. The final result will be a complete description of OHC electromechanical force production and its role in hearing.

DESCRIPTION (provided by applicant): A Biophysics Cell Membrane Probe (BCMProbe) is requested to expand biophysical studies on the membranes of living cells. The BCMProbe consists of optical tweezers integrated with an electrophysiology patch-clamp module and a standing wave microscope. The BCMProbe is a novel, unique instrument that can be used to modulate membrane mechanics as well as the magnitude of the electric field across it. At the same time it can measure the force produced by the membrane and the membrane current. Major users will exploit the instrument to further examine membrane electromechanics and actin bundling in nanoscale membrane tubes, measure charge movement associated with cell deformation and examine the self association as well as the mobility of integral membrane proteins. We will be able to expand our previous work that has demonstrated cell membranes capable of converting electrical energy directly into mechanical energy at acoustic frequencies. In addition we will to increase our understanding of mechanotransduction in Merkel cells, an epidermal mechano-sensory cell. The BCMProbe will be a new instrument at Baylor College of Medicine and its design is based on over 10 years of experience gained in constructing a previous model. The new instrument will be enhanced by super-resolution microscopy, electro-optic deflectors and complete system integration. The design is modular so that a variety of user specific experiments such as calcium imaging, fluorescence recovery after photobleaching or fluorescence resonance energy transfer may be easily accommodated. Our users have extensive expertise in optical approaches to membrane biophysics and mechanotransduction in sensory systems. The work will have implications beyond the hearing and somatosensory systems as it will reveal fundamental membrane mechanisms. These mechanisms have implications for understanding membrane transport and even learning and memory. While the obvious beneficiaries of the instrument will be the hard of hearing and individuals with touch disorders, solutions for other clinical problems will undoubtedly arise because every cell and the organelles within are enclosed by a membrane.