Robert E. Fyffe - US grants

Inhibitory mechanisms are known to be crucial in motor control, epilepsy, anaesthesia, and many other aspects of neural function. However, analyses of inhibitory mechanisms are complicated by uncertainties concerning the anatomy of inhibitory circuits and the location of inhibitory synapses on postsynaptic neurons. This project proposes to study the Renshaw inhibitory interneuron and its inhibitory connections with spinal motoneurons as a model to provide a better understanding of the mode of action of inhibitory circuits in the mammalian CNS. Identified Renshaw cells and target motoneurons will be intracellularly stained with horseradish peroxidase, using pipette microelectrodes, to directly determine the number and location of inhibitory synapses made by a Renshaw cell on a target motoneuron. This data will indicate whether Renshaw cells might act by inhibiting motoneuron firing in a global fashion (as a result of predominantly somatic locations of the synapses) or by more subtle inhibitory effects at specific dendritic locations, remote from the soma. The ultrastructure of identified Renshaw cell axons and terminals will be studied to determine their synaptic relationships, with identified motoneurons or other neural elements, and to provide insight into possible modes of junctional transmission at this synapse. Pharmacological analyses using blocking substances and analogues, applied both in vivo and in vitro, and immunocytochemical techniques, will be used to investigate the nature of the inhibitory neurotransmitter used by Renshaw cells.

DESCRIPTION (provided by applicant): Understanding how individual neurons in the mammalian central nervous system (CNS) communicate with each other requires detailed knowledge of the factors that regulate both synaptic transmission and the processing of synaptic signals by postsynaptic neurons (synaptic integration). In this project, we will address these issues by capitalizing on some key technical advantages offered by specific giant synaptic connections and postsynaptic neurons in central auditory pathways. We will study the properties of the same synapses and neurons in both normal and congenitally deaf animals, to gain insight into the role of activity in the development and regulation of synaptic strength and postsynaptic membrane properties in central pathways. Synaptic transmission and postsynaptic integration are inextricably linked - our study offers an extremely valuable opportunity to investigate both phenomena as comprehensively as possible in the same central neurons. Our aims employ patch-clamp recording of synaptic and membrane currents from calyceal axon terminals and their postsynaptic neurons, in parallel with immunolabeling and electron microscopy to localize key synaptic and membrane proteins and determine synaptic structure, and with computational methods to help understand the roles of specific ion channels in synaptic integration. The aims will test the hypotheses that altered auditory system activity during development (1) leads to changes in synaptic strength, structure, ion channel expression and neuronal excitability, and (2) disrupts the normal tonotopic organization of auditory circuits. The proposed research will use detailed, multidisciplinary approaches to study synaptic structure, function, and integration in a region of the mammalian nervous system that is highly advantageous for this purpose. The combined skills and resources of two experienced and productive laboratories will be brought to bear on these fundamental issues, which heretofore have been difficult to address in the mammalian CNS. The organization and geometry of auditory neurons and synapses allows electrophysiological and immunohistochemical results to be directly compared. The results to be obtained will be of general significance for understanding mechanisms of synaptic transmission and integration, and of clinical significance in understanding the changes that occur in the central auditory system under conditions of chronic sensory deprivation.

The long-term goal of this project is to define the structural and functional properties that underly the integrative functions and general excitability of spinal cord interneurons involved in the control of locomotion and posture in mammals. Their pivotal role(s) in motor control are well recognized, as is the notion that changes in interneuron excitability are likely to be contributing factors in a variety of locomotor disturbances consequent to disease or trauma. But the details of interneuron structure and function are lacking, particularly concerning the mode of action of descending control systems and cellular input- output characteristics. These gaps are a significant impediment in studies of the effects of spinal injury and the cellular/network mechanisms that underlie the resulting functional impairments. Although different classes of interneurons share some common features, there is striking diversity in terms of synaptology, receptor and ion channel expression, dendritic structure, and axonal projections. It is our hypothesis that these differences are functionally meaningful, and as a consequence they provide different classes of cells with a broad repertoire of potential responses to injury. We propose a comprehensive study of the structural and functional architecture of identified interneurons at the cellular level in the intact (uninjured) spinal cord. We will test the hypotheses that 1) dendritic structure and synapse distribution are two of the key factors that determine the relative amount of synaptic current, from dendritic synaptic sites, that reaches the cell soma and controls cell firing (i.e. input-output properties), and 2) that different classes of ventral horn interneurons are differentially innervated by descending motor control systems and display unique patterns of synaptic organization. This integrated proposal will focus on various well-defined interneurons in the adult cat spinal cord and will employ powerful combinations of quantitative light and electron microscopical, immunocytochemical, electrophysiological, and computational approaches. The new insights to be gained on the significance of dendritic structure and synaptic distribution, and definition of the overall cellular properties and organization of interneurons in spinal cord circuits, will help to establish the essential baseline for understanding the effects of spinal cord injury on interneurons.

In order to participate fully in the educational process, individuals with disabilities require innovative methods and technologies that are designed with a comprehensive understanding of learning with disability. However, few faculty have the interdisciplinary competencies to design and deliver pedagogically sound and accessible technology. The intellectual merit of this Integrative Graduate Education and Research Traineeship project is the establishment of an interdisciplinary concentration in technology-based learning with disability (research, curriculum, practicum) that spans multiple doctoral programs, including biomedical sciences, human factors/industrial organizational psychology, engineering, and computer science and engineering. The team will collaborate to train a new hybrid cohort of doctoral students who can bridge the gap between disability, assistive technologies and pedagogy of individualized learning. Interdisciplinary research projects available to IGERT trainees are grouped into three general areas: the basic nature of human performance (abilities and disabilities), the study of human-machine interactions (assistive technologies) and pedagogy (training systems development and access to learning). The concentration will also require a core of classes (such as science of learning, physiology of disability, legal and ethical aspects of disability), and a practicum that will be accomplished through service learning in the disabled community. Trainees will also be immersed in the culture of disability through program level activities. Additionally, to build community and a sense of purpose among the trainees, their concentration will be enhanced with the technological building blocks of a Universal Access Design Studio. The broader impact of this proposal is that it will improve learning for individuals with disabilities and will translate to increasing participation of individuals with disabilities in science and engineering. IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The goal of this alteration and renovation project is to maintain and improve the cage washing facilities at Wright State University. The present facility was constructed in two phases (1976 and 1986) resulting in a central dirty cage wash area with a single dirty cage wash area and two separate clean cage areas. The present flow of caging significantly increases the potential of contamination of clean caging and dramatically increases labor requirements. The facility also suffers deterioration from the use of acid and alkalie detergents and general wear. This application requests 1) the installation of a new rack washer to replace the existing 28 year old unit, 2) replacement of a gravity displacement autoclave with a large vacuum autoclave, 3) the addition of a steam generator to supply the autoclaves with "clean" steam, and 4) the relocation of the existing tunnel washer and autoclave to effect a true clean-dirty cage wash arrangement. This will minimize the potential of clean cage contamination from soiled cages, improve the flow of personnel and caging, and reduce overall operational costs and per diem charges. [unreadable] [unreadable] The renovation will also correct potential occupational hazards of slipping and acid exposure associated with the deteriorating flooring, scalding risk associated with the handling of hot and wet caging, and allergen exposure. This renovation is timely in that the University is initiating a program to completely renovate the physical and biological sciences buildings over the next 8 years. The present rack washer is located in a building scheduled for complete renovation in 2008. The loss of the rack washer would significantly impact the capability of the animal resources unit to sanitize caging directly affecting all ongoing research projects at the University. This proposal would permanently relocate the rack washer into the Health Sciences building ensuring continued cage sanitation and allowing the reclamation of the vacated adjacent cage wash area in the Biological Sciences building as animal housing or support rooms in the proposed renovation. As the only cage wash area for all University facilities, this project will directly impact all research using vertebrate animals at Wright State University. [unreadable] [unreadable] [unreadable]

Project 5: Peripheral nerve injury causes major alterations in neuronal activity, excitability, and synaptic organization in spinal locomotor circuits and motoneurons. The intrinsic membrane and integrative properties of neurons in the central nervous system depend on highly regulated expression and subcellular distribution of specific ion channels. Membrane ion channel clustering possibly represents an important and functionally significant mechanism for sequestering channels in specific membrane microdomains, presumably co- localized with other key signaling molecules, receptors, and ion channels. Voltage-gated Kv2.1 (delayed rectifier) channels are highly clustered at specific postsynaptic sites in motoneurons and the goal of this project is to elucidate mechanisms by which axon injury alters the subcellular distribution and clustering of membrane Kv2.1 channels, and to examine the functional relationships between altered channel distribution and intrinsic neuronal membrane properties. By studying the dynamics of Kv2.1 clustering in detail the first Specific Aim will test the hypothesis that motor axon damage, in vivo, causes Kv2.1 ion channels to become diffusely distributed in the somatic and proximal dendritic membrane following their dissociation from well defined large clusters. The second Specific Aim will consider the possible functional implications of any changes in Kv2.1 channel distribution, using electrophysiological analysis of intrinsic membrane properties in vivo. In parallel with PPG Projects 1 and 2, the quantitative confocal microscopic immunohistochemical analyses will extend over the complete time course of the motoneuron's response to axon injury, regeneration, muscle reinnervation (or lack thereof), and the loss and subsequent remodeling of identified excitatory and inhibitory synapses on the motoneuron soma. Complementary studies of channel localization and physiological role will also be performed for calcium-activated (SK2 and SK3) potassium channels and hyperpolarization-activated mixed cation channels (HCN1) that are abundantly expressed in spinal motoneurons and that underlie critical membrane properties that are altered after axotomy. This project will address fundamental gaps in our knowledge of ion channel organization in motoneurons and provide novel insight regarding the regulation of key membrane ion channels selected from the large number of channels that potentially make important contributions to cell excitability and integrative properties in normal and injured motoneurons (MNs). Importantly, this project helps to tie together two themes of this PPG, namely, the effects of injury per se and the activity-dependent regulation of cell excitability and will thus provide key insights into how postsynaptic excitability changes may contribute to altered network function following nerve injury.

PROJECT SUMMARY (See Instructions): Core B is essential to support the microscopic imaging, quantitative analysis, and peripheral nerve injury model used in this PPG. The Core is used extensively by all three projects and contributes directly to multiple goals of these projects. The scope and resources of Core B have been significantly expanded by addition or update of new instrumentation (e.g. an AMT digital camera for the electron microscope, funded by an Administrative Supplement to the PPG) to meet the needs of the PPG projects. A significant addition is the provision of expertise and technical help to provide a single model of peripheral nerve injury in rodents that is used by all 3 projects. This eliminates variability between projects in surgical technique and microinjection of tracers in specific muscles or nerves, and facilitates direct comparison of data between projects to aid in interpretation. The Core facility is directed by a highly experienced investigator and research administrator, assisted by three highly trained and qualified research associates. The Core provides all necessary training to members of the PPG groups for authorized access and use of Core instruments and facilities; PPG members have priority access to the facility. Services include training and guidance for use of instruments and analytical techniques, assistance with design and/or interpretation of experiments, hands-on performance of tissue processing and imaging as required, electron microscopy processing and imaging, and microsurgery and tracer injection. Significant resources include 3 confocal microscopes (one is a multiphoton instrument with electrophysiology setup), electron microscope suite, Neurolucida system, fluorescence dissecting microscope, cryostats, vibratomes and ultramicrotomes, cryosubstitution system, a fully equipped histology laboratory, access to a state-of-the-art suite for microsurgery in rodents, and four off-line workstations for image processing and data analysis. Data is securely stored, and each PPG PI has secure access to a PPG shared data Cloud (1.0 terabyte) where they can access their own and each other's data and communicate seamlessly with the data acquisition instruments in the Core.