William M. Roberts - US grants

The proposed experiments are directed at the long-term goal of understanding how neurons and other excitable cells regulate the complex patterns of electrical excitability and chemical sensitivity on their surfaces. Because ion channels are responsible for a cell's electrical properties and to a large extent determine how it will receive, integrate, and transmit information, a neuron must be able to synthesize the classes of ion channels appropriate for its functions, deliver them to specific positions on the surface, and remove them when their useful lifetimes expire. The proposed experiments address several aspects of these important cellular processes. Skeletal muscle fibers will be used because their surfaces are accessible for mapping ion channel distributions over large areas, and because they perform several functions common to excitable cells: they receive synaptic input, and initiate and propagate action potentials. Information gained from this study will be relevant to neurological disorders that involve altered excitability of neurons and specifically to disorders of neuromuscular transmission. The experiments will use twitch muscle fibers from frogs and garter snakes, and cultured embryonic chick muscle cells. Focal electrical recordings, using a patch voltage clamp technique will be used to map the spatial distribution of sodium channels on the cellular surface, with particular emphasis on the region of synaptic contact, where inputs are received and action potentials are initiated. The spatial resolution of the measurements will be about 1 muM, small enough to explore the distribution of channels within and between terminal boutons on snake fibers and close to synaptic gutters on frog fibers. Many of the experiments are designed to compare the organization of sodium channels and acetylcholine receptors that populate the postjunctional region of the muscle. They will determine whether the membrane is divided into microscopic domains that contain separate populations of ion channels, or whether the two types of channels intermingle. These results will be important to understand how channels are confined within the postjunctional membrane. Other experiments will explore the development of the neuromuscular junction, to determine whether the aggregation of these two types of channels around the neuromuscular junction occurs by similar of different mechanisms. Some experiments will measure the ability of sodium channels to migrate within the fluid membrane, an important factor in determining the channel distribution. These experiments attempt to resolve the discrepancies between two techniques that have given very different pictures of the mobility of sodium channels within the membrane. Finally, in vivo experiments will make the first measurements of the turnover rate and lifetime of sodium channels in adult muscles in living animals.

The cell surface receptor for the macrophage colony stimulating factor (CSF-1 or MCSF) is encoded by the c-fms proto-oncogene and is one of a family of growth factor receptors that triggers mitogenesis through an intrinsic, ligand-dependent tyrosinespecific protein kinase activity. This proposal outlines studies to identify and characterize the mechanisms that regulate transcription of the c-fms proto-oncogene. CSF-1R is primarily expressed on cells of the mononuclear phagocyte lineage and their committed progenitors, by leukemic myeloblasts, and by placental trophoblasts. Preliminary results indicate that transcription of the human CSF-1R gene originates from separate tissue-specific promoters. In the proposed studies, transcriptional regulatory sequences associated with each c-fms promoter will be inserted upstream of the chloramphenicol acetyltransferase (CAT) gene and tested for their ability to program gene expression in cell lines established from malignant placental trophoblasts and myeloid leukemic cells. Critical promoter/enhancer sequences responsible for transcriptional regulation of CSF-1R expression by human monocytes will be compared with analogous sequences associated with an alternative transcription origin used by placental trophoblasts. Nuclear proteins will be identified that influence CSF-1R expression through interaction with the alternative promoter/enhancer elements used by monocytic cells and placental trophoblasts. The studies outlined in this proposal will provide insights into the molecular basis of CSF-1R lineage-specific expression during placental development and normal or aberrant hematopoiesis. They also will clarify the recent observation of tandem linkage between the platelet-derived growth factor B-type receptor gene and the CSF-1R gene on human chromosome 5 - whether this positioning is purely coincidental or promotes novel regulation of CSF-1R expression.

WPC_ 2 B V P Z Courier 10cpi ? x x x , k x 6 X @ 8 ; X @ HP LaserJet III HPLASEII.PRS x @ , t 0 OpX @ 2 < Z L ! #| x Canon LBP-8III (Letterhead) CALB8IAD.PRS x @ 0 jFX @ Courier 10cpi Courier 10cpi Bold 2 X . ? x x x , l x 6 X @ 8 ; X @ l ? x x x , x ` w ; X > ' 9 >N ( ? 5 6N E 2 D < u | u | t D 5 t$ D E =& t =^ u y r= u t " s r 9320702 Roberts Salmons are well known for their ability to return to the stream in which they were born. The sensory basis for this remarkable behavior is olfactory: during a sensitive developmental period (smolt transformation), salmon imprint to distinctive homestream odors, and adults later use this odorant memory as a migratory cue to guide them back from the open ocean to their natal streams. How salmon imprint to homestream odors has remained an important but perplexing puzzle for over forty years. Proponents of olfactory imprinting have assumed that the olfactory memory for the home stream resides in the central nervous system. Dr. Roberts, however, proposes that olfactory memory is actually retained in the olfactory receptor cells themselves. The basis for his theory rests on two important findings. First, surges in plasma thyroxine levels mediate smolting, and second, olfactory imprinting coincides with this developmental metamorphosis. This small grant for exploratory research enables Dr. Roberts to use state of the art electrophysiological and neuroanatomical techniques to determine whether there are thyroxine induced change s in olfactory receptor neurons to specific odors. This basic research will have direct benefits to Pacific salmon by providing insight into how olfactory imprinting might be artificially manipulated. If thyroxine hormone can influence the ability of fish to imprint during a wider time frame than smolting, this finding could markedly advance conservation efforts aimed at transporting rapidly diminishing endangered salmon runs to healthier river systems. ***

Roberts, William M. IBN 94-10637 The ability to recognize familiar locations by their sights, sounds and smells is a common phenomenon. In salmon, who migrate great distances from fresh water streams to vast salt-water oceans, and ultimately, back to their home streams to reproduce, the ability to identify their home streams is crucial to species survival. But how is it that these fish recognize their home environments? One possible mechanism is the incorporation of familiar smells into memory which occurs during the parr smolt in salmon. During smolting, a physical transformation takes place as does a maturation of olfactory memory. Additionally, smolting is accompanied by a marked increase in thyroid hormone levels. Drs. Roberts and Takahashi will examine the role of thyroid hormones in the development of olfactory memory. Olfactory receptor cells born at the time of the parr smolt will be chemically marked and the effect of exogenous thyroid hormone treatment to enhance their survival will be determined. Moreover, structural changes in the olfactory bulb of the brain at smolting and modulation of those changes by thyroid hormones will be assessed. In addition to examination of the structural changes occurring in the brain at the time of smolting, functional changes in the sensitivity of the olfactory receptor cells to odorants will be examined. These studies have the potential to provide a breakthrough in understanding the physical basis of memory. Further, migration of salmon is of considerable commercial interest. This research has the potential to allow man to devise methods for guiding salmon to their birthplaces following novel pathways that may enhance the survival of the species.

Daniel Udovic DUE 9551950 U of Oregon Eugene FY1995 $39,083 Eugene, OR 97403 ILI - Instrumentation Project: Life Sciences Title: Promoting Investigative Learning in Biology Laboratories Using Computer-Based Data Acquisition Systems Teaching labs should be the intellectual catalysts of a curriculum, where students experience all aspects of the scientific endeavor, where they are challenged to confront their misconceptions about the natural world, and where they learn to construct explanations and meaning rather than find the "right" answers. In many undergraduate labs the equipment used to collect data acts as a barrier to student learning and participation. Often laboratory protocols force students to focus on the technical aspects of data collection while de-emphasizes the underlying scientific principles being presented. To be intellectually involved student must participate in investigations, that is, they must be involved in the design of experiments and the interpretation of results. The equipment requested in this proposal will make it possible to enhance student learning in biology teaching labs by replacing current analog devices, such as oscilloscopes and chart recorders with computer-based tools for data acquisition. The development of customized user interfaces and curricular materials designed to take advantage of the computer-based equipment will: emphasize the use of critical thinking skills by giving students the tools to quickly analyze their data; break down obstacles to learning by allowing students to focus important scientific concepts rather than instrumentation per se; and bridge the gap between teaching lab experiences and independent research opportunities by encouraging investigative lab activities. Materials necessary to equip two lab rooms (serving 48-60 students) with systems for electronic data acquisition and analysis will be purchased. The equipment will be general enough to be used in a wide variety of courses serving both biology majors and non-majors.

DESCRIPTION: The long-term objective of this work is to understand the mechanisms underlying calcium-mediated exocytosis of neurotransmitter during synaptic transmission. Chemical synapses are the targets of many toxins, therapeutic drugs, and drugs of abuse that affect the brain by altering synaptic transmission. Plasticity of chemical synaptic transmission underlies some forms of learning and memory, and disorders of specific neurotransmitter systems underlie a number of psychiatric, neurological, and neuromuscular diseases. A better understanding of synaptic transmission will thus advance human health. The proposed experiments will investigate the presynaptic physiology of hair cells from the frog saccules. Hair cells are the specialized sensory receptors of the auditory and vestibular systems. Their unique combination of anatomical and physiological properties make them ideally suited for studies of presynaptic mechanisms. These properties include: (a) individual hair cells can be isolated and studied in vitro; (b) their compact shape allows rapid control of the membrane potential at synapses, using the whole-cell and perforated-patch methods; (c) the presynaptic calcium current can easily be separated from other currents and recorded in voltage clamp experiments; (d) the presynaptic calcium concentration can be estimated from the activity of presynaptic Kca channels; (e) capacitance measurements can be used to monitor exocytosis; (f) known concentrations of exogenous substances can be rapidly added to the cytoplasm through the recording pipette. These features will allow a detailed analysis of the roles of calcium-binding proteins (calbindin-D28k and synaptotagmin) in synaptic transmission. The specific aims of the project are: (1) To test the hypothesis that calbindin-D28k influences short-range calcium signaling ion frog saccular hair cells. (2) To optimize the method for measuring rapid changes in membrane capacitance. (3) To test the hypothesis that depolarization-evoked increases in membrane capacitance are due to the fusion of synaptic vesicles with the plasma membrane at active zones. (4) To test the hypothesis that only the synaptic vesicles that lie alongside the rows of presynaptic calcium channels are available for immediate release, and that the maximum sustainable exocytotic rate is limited by the rate at which this release-ready pool can be supplied from other pool(s) of vesicles. Morphological studies using fluorescence microscopy and electron microscopy will be used to correlate the physiological measurements with synaptic structures. These studies will provide new, quantitative information about calcium signaling and neurotransmitter release that should be applicable to other synapses in the nervous system.

microscopy; nervous system; model design /development; lipids; informatics; biomedical resource;

This Shared Instrumentation Grant proposal requests funds to purchase a Zeiss LSM 510 NLO multiphoton laser scanning microscope, to be installed in the Bio-Optics Laboratory at the University of Oregon. This microscope will be used to advance ongoing NIH supported research in numerous laboratories in the Institutes of Neuroscience and Molecular Biology at the University of Oregon, including six major users who have described specific projects that will be greatly facilitated by the availability of a multi-photon fluorescence microscope. The two main benefits offered by this new technology are (1) the capability performing time-lapse microscopy on living specimens with greatly reduced photodynamic damage or bleaching of fluorescent labels and (2) the ability to obtain crisp images at depths of 100 mu m or more into a specimen. Five of the major users study the developmental biology of zebrafish (Judith Eisen, Charles Kimmel and John Postlethwait), Drosophila (Chris Doe), tunicates (John Postlethwait) and C. elegans (Bruce Bowerman). One user (William Roberts) studies synaptic physiology and calcium signaling in sensory receptors in the frog ear. The microscope will be available as a shared facility for use by life scientists at the University of Oregon.

DESCRIPTION (provided by applicant): The long-term objective of this work is to understand the mechanism of synaptic transmission in sensory receptor cells in the ear, eye and other sensory systems that use graded transmission at ribbon-class synapses. This specialized form of chemical signaling appears to be an adaptation to allow the transmission of information about small changes in sensory input that would be lost during conventional synaptic transmission that uses action potentials. The proposed experiments will investigate the physiology, anatomy and biochemistry of these synapses. The work on sensory receptors (hair cells) in the ear will use frogs and zebrafish as model species. The zebrafish work will also study photoreceptors and bipolar cells in the retina, and hair cells in the lateral line organs. All of these calls have ribbon-class synapses. These species were chosen for study because of the cellular and molecular tools that are available to answer fundamental questions in synaptic physiology. In the case of frogs, there is also a wealth of information already available upon which to build. During the past 10 years, several laboratories have developed techniques that allow detailed electrophysiological analysis of synaptic transmission using tight-seal voltage clamp to measure small changes in membrane capacitance to observe synaptic transmission on a millisecond time scale. This physiological method will be used in conjunction with electron tomography and recently developed membrane tracer dyes to study the ultra structure of the synaptic vesicle cycle ribbon synapses. The goal is to test several key hypotheses concerning the function of the synaptic "ribbon", the prominent anatomical feature for which these synapses are named. The project will also focus on two major proteins (calretinin and parvalbumin 3) that are believed to serve central roles in synaptic transmission in these cells by capturing and transporting calcium ions away from the synapses. The project will investigate the important biochemical properties that determine how fast these proteins bind calcium, how much calcium they can sequester, and how fast they can diffuse within the cell. These properties are central to understanding synaptic transmission in hair cells, and have a wider relevance to the proposed function of these and related calcium-binding proteins in protection from calcium overload during strokes and other brain injuries. The genetic and molecular tools developed by zebrafish researchers during the past decade will allow a direct test the function of a protein (Ribeye) that has recently been identified as a major component of the ribbon.

Address; Blood Coagulation Factor IV; CRISP; Ca++ element; Calcium; Cell membrane; Chemical Synapse; Chromosome Pairing; Class; Coagulation Factor IV; Computer Retrieval of Information on Scientific Projects Database; Corti Cell; Cytoplasm; Cytoplasmic Membrane; Dependence; Dimensions; Electrons; Exocytosis; Factor IV; Funding; Goals; Grant; Hair Cells; Institution; Investigators; Lead; Location; Math Models; Measures; Mediating; Modeling; NIH; National Institutes of Health; National Institutes of Health (U.S.); Negative Beta Particle; Negatrons; Nerve Transmitter Substances; Neural Transmission; Neurotransmitters; Numbers; Organelles; Pb element; Physiologic; Physiological; Plasma Membrane; Research; Research Personnel; Research Resources; Researchers; Resources; Source; Synapses; Synapsis; Synapsis, Chromosomal; Synaptic; Synaptic Transmission; Synaptic Vesicles; Testing; United States National Institutes of Health; VESCL; Vesicle; Work; body map; ear hair cell; heavy metal Pb; heavy metal lead; mathematical model; mathematical modeling; plasmalemma; presynaptic; synapse function; synaptic function

Technical Abstract

In this proposal we request funds to purchase a state-of-the-art optical system for performing Near-field Scanning Optical Microscopy (NSOM) with spectroscopic capabilities. The promise of NSOM lies in its ability to provide wavelength-dependent optical imaging in spatial regimes well below the diffraction limit; its imaging and spectroscopic capabilities offer structural and compositional information at a spatial resolution much higher than traditional optical microscopy. The NSOM therefore comprises a crucial bridge between optical diffraction-limited systems and higher-spatial-resolution non-spectroscopic techniques such as atomic force microscopy and electron microscopy. Characterization of materials in this spatial regime is of paramount importance as the shift from "micro" to "nano" continues. The system will be configured to ensure an immediate contribution to research programs based at the University of Oregon in fields ranging from nanophotonics to biological sciences. The NSOM capabilities will be exploited to extend ongoing research on coherent optical phenomena in highly confining metallodielectric nanostructures. The requested system is ideally suited for investigation of plasmon localization, nonlinear plasmonics active nanoplasmonic systems. As NSOM has also been shown to be of great usefulness in the characterization of organic and biological materials, it will be used by several research groups with interests in these fields. In one project, it will be used to investigate the spatial organization of ion channels at synaptic active zones in receptor cells. In a separate research group the NSOM will facilitate spectroscopic imaging of surface-adsorbed biomolecules at the submicron level. In another project, charge transport in photoconductive organic semiconductors will be investigated at the single molecule level. As part of the multi-user Surface Analytical Facility at the University of Oregon, the NSOM would be optimally positioned to impact a broad range of emerging research programs, both within the university and outside, as well as expand the current facility user base.


Non-Technical Abstract

This proposal addresses the acquisition of a specialized high-resolution microscopy system, capable of providing information about the structural and compositional characteristics of minute structures. A large variety of systems may be studied with this instrument; some examples include very small metal particles , biological cells, light-emitting molecules and thin oil slicks on water surfaces. What unites all of these systems is their size scale -typically about one half of one thousandth the width of a human hair, and often even smaller than that. Conventional optical microscopes such as those in high school science labs cannot reveal spatial features of objects at such small size scales. This limitation is inherent to all commonly available optical systems which employ light and lenses for visualization and magnification of images. The smallest spatial feature which may be distinguished using these instruments is about one half the wavelength of visible light - approximately five to ten times larger than the systems we are interested in studying. However, if instead of a lens a minute aperture (even smaller than our smallest object!) is placed at very high proximity to the sample, imaging of much finer features is possible when the aperture is illuminated with visible light. Such a system will allow students and researchers at the University of Oregon and its affiliated Institutions to elucidate the characteristics of novel systems such as the smallest light emitting structures known to nature, fabricate ultra-compact electronic and optical devices, understand how chemical contaminants interact with their immediate environment, and study biological cell structure and functionality with unprecedented optical resolution.

Project Summary The nematode worm Caenorhabditis elegans has proven valuable as a model for many high-impact medical conditions. The strength of C. elegans derives from the extensive homologies between human and nematode genes (60-80%) and the many powerful tools available to manipulate genes in C. elegans, including expressing human genes. Researchers utilizing medical models based on C. elegans have converged on two main quantifiable measures of health and disease: locomotion and feeding; the latter is the focus of this proposal. C. elegans feeds on bacteria ingested through the pharynx, a rhythmic muscular pump in the worm?s throat. Alterations in pharyngeal activity are a sensitive indicator of dysfunction in muscles and neurons, as well as the animal?s overall health and metabolic state. C. elegans neurobiologists have long recognized the utility of the elec- tropharyngeogram (EPG), a non-invasive, whole-body electrical recording analogous to an electrocardiogram (ECG), which provides a quantitative readout of feeding. However, technical barriers associated with whole- animal electrophysiology have limited its adoption to fewer than fifteen laboratories world-wide. NemaMetrix Inc. surmounted these barriers by developing a turn-key, microfluidic system for EPG acquisition and analysis called the the ScreenChip platform. The proposed research and commercialization activities significantly expand the capabilities of the ScreenChip platform in two key respects. First, they enlarge the phenotyping capabilities of the platform by incorporating high-speed video of whole animal and pharyngeal movements. Second they develop a cloud database compatible with Gene Ontology, Open Biomedical Ontologies and Worm Ontology standards, allowing data-mining of combined electrophysiological, imaging and other data modalities. The machine-readable database will be compatible with artificial intelligence and machine learning algorithms. It will be accessible to all researchers to enable discovery of relationships between genotypes, phenotypes and treatments using large-scale analysis of multidimensional phenotypic profiles. The research and commercialization efforts culminate in an unprecedented integration of genetic, cellular, and organismal levels of analysis, with minimal training and effort required by users. Going forward, we envision the PheNom platform as a gold standard for medical research using C. elegans.