David K. Ryugo - US grants

Our overall objective is to understand the mechanisms of signal processing in the auditory nervous system. The cat cochlear nucleus provides a model system for studying how sensory nerve inputs produce output discharges in second order neurons. In a general sense, the function of the cochlear nucleus is to receive incoming auditory nerve discharges, modify the message and distribute the resulting output signals to higher centers in the brain. How auditory information is subsequently processed by the brain will be heavily dependent on events in the cochlear nucleus. Because functional properties must ultimately be based on anatomy, we have developed techniques whereby single auditory nerve fibers can first be electrophysiologically characterized by recording with micropipettes inserted into the axon, and then be labelled by intracellular injections of horseradish peroxidase (HRP) through the same pipettes. After histological processing, each labelled neuron can be reconstructed from serial sections from its peripheral ending in the cochlea to its central ramifications in the cochlear nucleus. Since the HRP reaction product is electron dense, these identified neurons can ultimately be examined with electron microscopy in order to determine the nature of their synaptic connections. These methods for staining and studying single neurons after characterizing their physiological response properties will allow us to describe structure-function relationships at the cellular level. The compilation of these data should help to generate a new level of understanding for mechanisms of signal processing, and will set the stage for studying the consequences of cochlear and central pathology on the cochlear nucleus. The possibility of practical prosthetic devices that successfully bypass nonfunctioning cochleas should insure continued interest in studies of stimulus coding at the level of the auditory nerve and cochlear nucleus.

The cochlear nucleus is the terminus for all incoming auditory information to the brain and the origin of all central auditory pathways. In a general way, the role of the cochlear nucleus is to recode signals from the auditory nerve and to distribute the resultant messages to higher centers. The long term goal of this research is to understand the structural basis by which neural activity from the auditory nerve is "processed" in the cochlear nucleus. This effort requires a thorough knowledge of the synaptic organization in the cochlear nucleus because the coding process is hypothesized to be dependent on definable features of the relationship between pre- and postsynaptic neurons. The present proposal will apply intracellular recording and staining methods to reveal the structural and functional character of individual neurons, tract-tracing methods to reveal the source and distribution of input projections, and immunocytochemical techniques to identify molecules associated with neurotransmitter cell classes. These data will have direct relevance to ideas about excitation and inhibition in the cochlear nucleus and about how neural circuits act to shape the coding process.

DESCRIPTION (Investigator's Abstract): In the mammalian auditory system, information pertaining to hearing enters the brain by way of the auditory nerve. The axons of two types of primary neurons, type I and type II spiral ganglion cells, compose the nerve and terminate in the cochlear nucleus. In a general way, the function of the cochlear nucleus is to receive incoming auditory nerve activity, to preserve or transform the signals, and to distribute outgoing activity to higher brain centers. Our long-term objectives are (1) to describe the neuronal circuitry in the cochlear nucleus and (2) to understand the mechanisms underlying the early stages of acoustic signal processing in the brain. Knowledge of these issues will depend substantially on the organization of auditory nerve input to the cochlear nucleus. Intracellular recording and staining methods will be used to label individual type I auditory nerve fibers. The marking of single fibers with horseradish peroxidase (HRP) after first characterizing their response properties allows a direct comparison between a fiber's response features, its axonal and synaptic morphology and its connections with other neurons. Because of the difficulty in recording from type II auditory nerve fibers, we will concentrate our analysis on their light and electron microscopic features and compare them to those of type I fibers. Type II fibers will be labeled using extracellular injections of HRP in the auditory nerve or spiral ganglion. We will test the hypothesis that type II fibers exhibit systematic differences in their synaptic morphology and connections compared to type I fibers. The proposed studies should generate new information regarding the anatomical foundations of stimulus coding and neural circuitry in the auditory nerve and cochlear nucleus. The data will also have relevance to broader issues in sensory neurobiology and could be especially important for the design of prosthetic devices that replace nonfunctioning cochleas or auditory nerves.

[unreadable] DESCRIPTION (provided by applicant): Perception of sound is attributed to the pattern of neural activity within the central auditory system. The auditory system is typically defined as a series of structures that are connected directly or indirectly to the cochlea. The significance of a sound, however, depends not only on spectral and temporal characteristics as mediated through the auditory system, but also on the past history and behavioral state of the animal. In other words, sound processing should also include non-auditory systems that would convey sensorimotor functions, emotion, learning, and memory. In this application, we plan to study the nature of non-auditory projections to the cochlear nucleus in order to gain insight into how different systems influence our perception of sound. We propose that the granule cell domain of the cochlear nucleus represents a key site for the integration of mutimodal influences. Preliminary data reveals that somatosensory, vestibular, pontine, reticular, and descending auditory projections access the granule cell domain. We will use anterograde and retrograde tract tracing methods in conjunction with light and electron microscopy to identify projecting neurons and their circuits, immunocytochemical staining procedures to reveal the chemistry of the different terminals, and electrophysiological recording techniques to characterize the response properties of neurons projecting to this region. We will apply nanoparticle technology to explore the use of biodegradable polymers with the goal of creating a new line of neuronal tracers for this study. The possibility of attaching ligands to the surface of the particles could enhance tracer uptake for the selective targeting of specific families of neurons. The data from this research will provide new knowledge on the synaptic organization of a highly integrative structure in the auditory system, and should yield insights into how diverse neural systems shape the coding process for hearing. As we learn about how the somatosensory system modulates hearing, we will gain insight into somatic tinnitus that could contribute to the design of treatment strategies and a better understanding of tinnitus in general. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): [unreadable] We are requesting funds to purchase an electron microscope to replace our current 17-year-old one that has become increasingly unreliable. The proposed microscope will be used by a group of 11 users primarily from the Departments of Otolaryngology-Head & Neck Surgery and Ophthalmology at the Johns Hopkins University School of Medicine. A total of 13 projects will be supported by 8 RO1, 2 KO8, 1 K23, and several nonfederal grants. The purchase of this microscope is crucial for the continued progress of research supported by NIH programs. The breadth of the projects is broad but unified by a foundation in structural neurobiology. Projects include (a) studies of the synaptic organization and plasticity of the mammalian auditory system; (b) histopathologic studies of the human eye; (c) ultrastructural examinations of sensory receptors and associated structures of the eyes and ears in normal, pathologic, and/or genetically altered animals; (d) localization of molecules (cytoskeletal and synaptic proteins, growth factors, or ion channels) in eyes and ears that pertain to normal function; (e) identification of growth factors that enhance photoreceptor growth; and (f) gene transfer experiments involving factors that influence laryngeal nerve growth, reinnervation, and muscle development. Conceptual and technical approaches utilized by the different research groups emphasize the common themes that link these various projects. Our work is highly dependent upon electron microscopy, and the proposed purchase will provide its users with a reliable instrument that is equipped with a number of advanced features that are currently unavailable anywhere at our institution. Award of this instrument will have an immediate and positive effect on the productivity of these projects. It will serve to catalyze interdisciplinary discussions among users, provide a valuable teaching tool for our trainees, and enhance the progress of biomedical research on hearing, vision, and voice. [unreadable] [unreadable]

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