George A. Spirou - US grants

The goal of this project is to understand the roles of local feedback systems to the cochlear nuclei in shaping the neural representation of sound for processing by higher brain centers. Three local feedback systems which reside in the brainstem have been identified. These are: (1) the system controlling the middle ear muscles, including pathways subserving the middle ear muscle reflex; (2) the system which projects from the superior olive to both the cochlea and cochlear nuclei, the so-called olivo-cochlear system; and (3) the system which projects from the superior olive exclusively to the cochlear nuclei. The cells which give rise to the latter two systems are scattered in groups called peri-olivary nuclei (PON), which surround the principal cell groups of the superior olive. Cells of the PON receive direct projections from the cochlear nuclei, and project to diverse auditory brainstem nuclei. One cell group, the lateral nucleus of the trapezoid body (LNTB), consists of cells which project heavily to the ipsilateral cochlear nucleus. Therefore, the LNTB represents a well segregated part of the local feedback to the cochlear nuclei and has a relatively simple organization. This project is a focused study of the LNTB and may reveal principles which can aid in understanding the roles of other descending projections to the cochlear nuclei. A description of the extent to which the LNTB is influenced by input from the cochlear nuclei, as opposed to inputs from higher brain centers, will be the aim of many experiments in this proposal. The experiments are guided by the hypothesis that feedback projections play a role in preserving the representations of sound in the presence of noisy backgrounds, and can work by the mechanism of affecting lateral inhibition of cells of the cochlear nuclei. The experiments are divided into two groups. The first group of experiments is designed to determine the anatomical organization and physiological properties of LNTB neurons. The second group of experiments test the hypothesis of LNTB function. The proposed experiments use a combination of in vitro (brain slice) and in vivo electrophysiological and anatomical techniques that are already in use in the laboratory. The combination of preparations will bring greater experimental control and flexibility to bear on investigation of LNTB functions and mechanisms. These experiments may reveal neural mechanisms of signal extraction in a noisy background, which may also provide information important for improving designs of prosthetic devices that replace nonfunctioning parts of the auditory nervous system.

EXCEED THE SPACE PROVIDED. The initial COBRE grant established the Sensory Neuroscience Research Center (SNRC) with a multidisciplinary nucleus of independent investigators at West Virginia University. This COBRE support stimulated institutional commitments for new tenure-track salary lines and the construction of modern laboratory space for SNRC expansion and enabled SNRC investigators to obtain R01 funding. We request a second phase of COBRE support to complete the development of a self-sustaining research enterprise, and thereby increase the research capacity of our institution. We propose 4 specific aims. Aim 1 is to achieve a critical mass of independently funded scientists. We have grown from 4 to 10 WVU investigators during the current funding period. In this application, 5 of these scientists have proposed new projects to aid transition to independent R01 funding. With proposed grant support we will further expand to 14 faculty members by recruiting established scientists, especially clinician investigators to broaden our studies of mechanisms of sensory disorders. Aim 2 is to advance understanding of sensory systems by fostering new collaborations among faculty, thereby integrating new investigators into the Center. Recent and projected recruits extend the collective scope of our work and add new technical expertise that underlies unique opportunities for scientific interaction. Aim 3 is to expand institutional research infrastructure through expansion of core facilities. Support is requested for SNRC core facilities for research and administrative activities, personnel to administer these cores, and partial support for institutional core facilities that are central to SNRC research efforts. Aim 4 is to transition the SNRC to independent status. With further COBRE support, we will obtain a strong base of R01 funding that underpins applications for research core and program project grants. Our Health Sciences Center has implemented a strategic research plan to provide resources for infrastructure and faculty recruitment and increase the number of qualified graduate students to train in our laboratories and support our research programs. Five research projects encompassing four sensory systems (auditory, olfactory, somatosensory, and visual) are proposed. These projects reflect the multidisciplinary approach of SNRC, which includes the comparison across sensory modalities and multiple levels of analysis (molecular to systems), in species ranging from invertebrates through humans.

This application seeks to study molecular mechanisms underlying the formation of orderly (tonotopic) connections in the auditory brainstem. The pathway to be studied is the projection of bushy cells of the anteroventral cochlear nucleus into the medial nucleus of the trapezoid body (MNTB). The project has two aims. First, the time course and growth patterns of innervation of MNTB will be studied using time-lapse imaging of fluorescence labeled axons. Second, candidate molecules that might be involved in axon guidance and target identification will be screened from core samples of MNTB. The spatial pattern of expression of these candidate molecules will be studied in organotypic slice cultures using in situ hybridization and immunocytochemistry.

DESCRIPTION (provided by applicant): A critical cue for localizing sound is the difference in the time of its arrival at the two ears, which is first analyzed in a brainstem structure called the medial superior olive (MSO). MSO neurons are activated when excitatory inputs from the two ears arrive in coincidence, but they also receive massive inhibitory inputs whose roles in MSO function have not been elucidated. We propose that two distinct groups of inhibitory neurons operate in complementary fashion to limit the duration of the coincidence detection window and preserve sensitivity to interaural temporal differences of MSO neurons. One cell group, which may synchronize its activity to excitatory inputs in order to shorten their duration, is active at low sound levels and uses glycine as its neurotransmitter. The second cell group, which generates asynchronous, tonic hyperpolarization in the MSO cell and thereby requires exact coincidence of excitatory inputs to reach action potential threshold, is active at high sound levels and uses GABA and glycine as its neurotransmitters. Aim 1 will study the structure and termination patterns of these inhibitory circuits. We will test the notion that synchronous inhibition is tonotopic and matched to the pattern of excitatory innervation but that asynchronous inhibition is diffuse and crosses tonotopic axes of the MSO. In Aim 2 we will measure the synchronization of inhibitory neurons to sound through extracellular single unit recordings of their activity. Aims 3 and 4 will test the role of inhibition in coincidence detection and in the representation of simultaneous sound sources in the population of MSO cells. Single unit activity of MSO neurons will be recorded in the absence and presence of iontophoretically applied antagonists of glycinergic and GABAergic neurotransmission. By defining biological mechanisms underlying sound localization, the proposed research will contribute to better strategies for hearing preservation following damage to the auditory system, such as through design or activation of cochlear nucleus prostheses, and for the design of speech recognition systems used in complex acoustic environments

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. ULTRASTRUCTURAL BASIS FOR SYNAPTIC VESICLE RECYCLING IN THE CALYX OF HELD The calyx of Held and other large nerve terminals of the auditory brainstem are key elements of sound localization circuitry. Our goal is to reveal structural transformations and cellular communication that characterize contact of the calycigenic growth cone with its target and the early stages of synapse assembly and stabilization at large nerve terminals in the auditory brainstem. Our central hypothesis is that competition among calycigenic inputs precedes expansion of the terminal over the cell body to form a calyx. This hypothesis is based on work from our laboratory that reveals rapid formation of the calyx in mice between postnatal days (P)2 and P4. The calyx contains hundreds to greater than two thousand active zones, depending upon the species, many of which are located nearby specialized organelle complexes termed mitochondrion-associated adherens complexes (MACs). MAC structure had been described previously using standard transmission electron microscopy (sTEM), which revealed filaments tethering the mitochondrion to a punctum adherens that links the pre- and postsynaptic membranes. Confocal fluorescence imaging and electron tomography are being employed to study medial nucleus of the trapezoid body (MNTB) cell innervation during P0[unreadable]P4. This time period precedes and overlaps the formation of immature, cup-shaped calyces that envelop the MNTB cell body. These experiments will pinpoint time periods during which mono-innervation is established between pre- and postsynaptic partners and highlight structural and functional differences that may predict winning and losing inputs. In addition, we will describe structural features of mature calyces that support high-rate neurotransmission that is characteristic of this terminal. Because of the size of the calyx-MNTB contact and the desired high resolution of the reconstructions, serial volumetric imaging of domains of cells will be required. This project will require high-resolution, wide-field, large-area digital recording of images for electron tomography.

Abstract Not Provided

Abstract not provided.

developmental neurobiology; synaptogenesis; intercellular connection; neural transmission; growth cones; calcium flux; auditory pathways; auditory nuclei; clinical research; fluorescent dye /probe; laboratory mouse; calcium indicator; single cell analysis;

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We structured our grant such that the administrative core supports the administrative office, startup packages for new faculty, phase-out budgets for successful projects, and equipment cores that are administered within the SNRC (biochemistry, histology, molecular biology and tissue culture, imaging) or by the Health Sciences Center (transgenic rodent, center for advanced imaging, electron microscopy), summer undergraduate research fellows and attendance of SNRC members at the annual WVU Center for Neuroscience retreat. As new faculty members join the COBRE, their budgets can be assigned initially as subprojects through this core to provide for immediate purchase of key startup resources for their laboratories. Subsequently, their research proposals are presented to our external advisors for approval and their budgets are assigned as individual tasks in our accounting system. In this section we describe activities in all of the listed areas during the past year. Ms. Angela Harrison, MPA, has been a very able Administrator for the SNRC. Ms. Harrison has proven extremely adept at conducting all of the operations of the SNRC, including accounting for the CoBRE grant, assisting investigators with submission of grant applications to the NIH, and managing SNRC activities such as visits by our external advisors and biweekly works-in-progress meetings. She has been the liaison between SNRC faculty and the lead personnel for the architectural firm, construction contractor and WVU planning personnel during the construction of our new laboratory space. She has relocated out of state, and completed her employment at the end of November, 2008. Her administrative duties are now shared with Stacey Malecky, MBA, who is also the Administrator for the Center for Neuroscience, the umbrella center under which the SNRC is organized. We are in the process of hiring a second person in our office, who will assist Ms. Malecky with administration of both centers. Both Ms. Malecky and her assistant will be supported 50% by the CoBRE grant. During the past year we have been unsuccessful in recruiting a specialist in regeneration or development of sensory systems and have developed a better approach to make this recruitment. We were targeting an established scientist in order to bring additional senior leadership and mentoring skills into our group. In order to address this issue, we have recently received a commitment to increase the value of the startup package we can offer. This startup package will be comprised of both institutional and CoBRE funds. Furthermore, we have received a commitment to recruit a second senior faculty member, also with a competitive startup package. That second startup package will also consist of CoBRE and institutional funds. Essentially we have leveraged the CoBRE startup into a second senior faculty position. We think that recruitment of two positions simultaneously will increase the attractiveness of each position, because we can demonstrate the possibility for immediate opportunities for collaboration with another senior scientist. One of these new faculty will be appointed in the Department of Neurobiology and Anatomy, as originally planned, and the other will be appointed in the Department of Otolaryngology. We anticipate that the opening of our new research building provides additional incentive for recruitment of high quality faculty. It is important to emphasize that our faculty growth has been entirely through filling of tenure-track, state funded positions. We have recruited faculty strictly from outside of the university into these positions. It is strategically important, we think, that a small institution continue to refresh its ideas and approaches to the conduct of scientific investigation. Drs. Visvanathan Ramamurthy and Sepideh Zareparsi continued to receive startup support via the COBRE grant. Dr. Ramamurthy has already received R01 funding for his research on retinal degeneration. During the past year, he also became the PI for the grant of Dr. Janet Cyr, a former SNRC member who left the group this year to become a program administrator at the NIH. Dr. Ramamurthy is now developing gene therapy approaches to treat retinal degeneration, which will form the basis for another extramural grant application. Dr. Ramamurthy exclusively uses animal models. The necessary ACUC approvals are included among the submitted documentation. Dr. Zareparsi is proceeding with human subject enrollment in her genetic studies of glaucoma, and submitted applications to two eye research foundations. One application received a competitive score and awaits a funding decision and the other is pending review. Please note that Dr. Zareparsi exclusively uses human subjects in her work. The necessary IRB and subject enrollment forms are included among the submitted documentation. Dr. Agmon continued to receive support during the past year for a postdoctoral fellow to maintain his competitiveness for an upcoming renewal. Dr. Agmon was a junior investigator who achieved R01 funding. This is the last year of support for Drs. Agmon and Zareparsi. Dr. Ramamurthy will maintain a small budget during the coming year. Administrative support for SNRC core facilities remains integral to our ability to conduct modern research. All of our facilities are used heavily. As an example, the confocal microscope is used increasingly each year and, although it is dedicated to SNRC researchers, investigators must plan experiments and reserve time up to one week in advance. The machine is used consistently outside of normal working hours. Our labs increasingly employ in vitro and in vivo imaging techniques, and we see the growing importance of imaging in our work. The collaboration that we instituted nearly two years ago with Feruz Ganikhanov, PhD, a faculty member in the Physics Department, to construct a non-linear optical imaging system is now functional. We have shared in the investment to create this facility, described in greater detail in other sections. This system has immediately provided us with the capability to conduct 2-photon imaging experiments. We have created an imaging resource that is cutting-edge, and available in only a small number of research institutions. We relocated this facility from the physics department to our new research building in December, 2008. It also serves as an important laboratory startup resource for recruitment of new faculty. The CoBRE grant also supports Health Sciences Center core facilities. The Transgenic Rodent Facility is supported via 50% salary coverage for the facility technician. The director of this facility is Dr. Peter Mathers, an SNRC faculty member. The electron microscope service contract is supported by the CoBRE grant. SNRC faculty are the heaviest users of this instrument. Discussions are underway with our Health Sciences Center administration that will result in expanded staffing of the electron microscopy facility. The electron microscopy suite will be moved across the hall from its current location to make room for expansion of the animal quarters. We are currently designing the new suite, which will house the electron microscope and a darkroom. We anticipate completion of the new suite by April, 2009. The Center for Advanced Imaging has been supported indirectly via the research project on the CoBRE grant held by Dr. Lewis. Our mentoring program remains strong. We will focus upon our grant mentoring committees of 2-3 people during the coming year so that our remaining junior investigators, Drs. Lewis, Sokolov and Zareparsi, can obtain NIH R01 funding. We also will expand the Summer Undergraduate Research Program (SURI) from 12 to 15 students during the summer, 2009, and request support for 7 of these students from the CoBRE grant. This expansion reflects participation by additional members of the Center for Neuroscience and an increased effort to recruit students to our graduate programs We conducted our most recent on-site meeting for the External Advisory Panel in September, 2008. We were hoping to combine this meeting with a symposium on sensory neuroscience to celebrate the move into our new laboratory space in the new research building. However, our move into the new building was delayed until November, so we are considering new dates during late summer to early fall, 2009, to hold a symposium. We currently have four external advisors and plan to add one member during the coming year. A very noteworthy event from the past year is completion of a new research building that houses the SNRC on its top (fourth) floor. The floor occupies 25,000 gross sq ft and has room for 10 laboratories. Currently 5 SNRC investigators are located in the new building, so the institution has programmed space for us to accomplish our projected expansion. This laboratory space will facilitate recruitment of established, funded investigators to join the SNRC.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our long-range goal is to reveal structural transformations and cellular communication that characterize contact of the calycigenic growth cone with its target and the early stages of synapse assembly and stabilization at large nerve terminals in the auditory brainstem. Our central hypothesis is that competition among calycigenic inputs precedes expansion of the terminal over the cell body to form a calyx.

This project is designed to meet the directives of the CoBRE phase III Transitional Award and will (1) further develop collaborative science and enhance training capabilities for faculty, postdoctoral and graduate trainees and undergraduate students and (2) support modern core facilities and continuously evolve them to provide modern technologies for collaborative research. We will continue existing, effective programs in our Center for Neuroscience for faculty mentoring, support for grant application and management, collaboration and interaction among all CN members, and a summer undergraduate research internship. We will support an institutional core facility director with broad responsibility for core facility oversight, financial management, efficiency and accessibility and advanced technology assessment. Five institutional cores will be supported, which have received support through earlier phases of the CoBRE grant: Transgenic Animal; Genomics; Advanced Imaging; Light and Electron Microscopic Imaging; Tissue Processing and Analysis. Each core is staffed with expert directors and three cores operate with doctorate level laboratory managers, ensuring high levels of expertise for experimental consultations with core users. The Advanced Imaging and Light and Electron Microscopic Imaging cores operate as technology discovery cores through collaborative involvement of laser, magnetic resonance and nuclear physicists who conduct research and assist with core management. Several core facilities are unique in our state, and support basic and translational research.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This pilot project mechanism is modeled after internal grant mechanisms at WVU that are peer-reviewed and require adequate progress and external grant submissions after completion. The CoBRE phase III will support three multi-disciplinary and highly innovative projects at $50,000 per year in year 1, followed by two projects at $50,000 per year in year 2 and one project per year in subsequent years. Grants can be renewable for up to one year, but only on a competitive basis, with significant progress expected in year 1 for renewal. Multi-investigator applications that work across several disciplines to answer significant biological questions in a unique manner will be given highest priority. Applicants are expected to utilize technologies available in the CoBRE-supported core facilities. Review of applications will be performed by CN investigators, along with other WVU investigators with relevant expertise, who have previous external grant review experience. Progress reports will be evaluated at approximately the six-month progress time by the CoBRE PI and external advisors and a subset of members of the grant review committee.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The administrative core supports the administrative office, summer undergraduate research interns (SURI Program), the annual WVU Center for Neuroscience (CN) retreat, the bimonthly Breakthrough Blitz, and biotechnology workshop/seminars.

DESCRIPTION (provided by applicant): The nervous system is a complex arrangement of neurons, their axonal projections, glia and vascular supply, yet our concepts about information processing are heavily neuron-centric. This bias results from the efficiency of standard anatomical techniques to map synaptic connections between neurons relative to other physical interactions among neural and non-neural cells. Spatial organization of neuronal groups and glia networks is indicated in many studies, but spatial organization and interactions have not been studied with an integrated approach. We will employ a new technology, serial block-face scanning electron microscopy, to reconstruct, at high resolution, all cellular elements of large tissue volumes to identify new principles in nervous system cellular organization. We will tap into human visual-cognitive capabilities to understand spatial order by developing procedures, implemented in a software user interface, to display combinations of cellular elements and vascular structures in 3-D virtual reality environments. Identification of structural features that define 3D organization of tissue will facilitate investigation of large-scale tissue volumes by othr investigators as high-throughput, high resolution studies of brain structure become more common.

The cochlear nucleus is the gateway for central nervous system processing of auditory information in mammals. It has been proposed that parallel processing channels are set up in the CN, and these form the basis for further computation at higher stations of the auditory system. Despite decades of study, enumeration of CN cell types is incomplete and CN circuitry is described only superficially. In neuroscience generally, classification and naming of neurons has relied primarily upon qualitative approaches based upon human observational capabilities. We have implemented and in some cases developed novel high-throughput and unbiased techniques for labeling, segmenting and classifying neurons in 3D, generated from large-scale electron microscopy image volumes. We propose to deliver a nanoscale map, or connectome, of the mouse CN with enumerated and localized cell types and their synaptic connections. This effort is unbiased because all neurons will be sampled. To achieve this goal, we bring together four parallel modes of tissue analysis for neuron classification: morphology, connectivity, molecular identity and function. We propose that connectivity analysis will define long-proposed parallel processing circuits that will be tested functionally using realistic biophysical models of identified cell types. Notably, the cochlear nucleus contains both amorphous and layered organizations of cells, which serve as templates for all other brain regions. By investigating the fundamental structure of this sensory center, we will establish principles of neural computation and methods for structural and functional phenotyping that will apply to other brain regions regardless of their particular neural architecture.