Michael E. Dailey - US grants

DESCRIPTION: The long term goal of this research program is to identify cellular and molecular mechanisms that regulate the formation and plasticity of synaptic connections in CNS tissue. The present proposal will focus on the development of the major postsynaptic specialization in the CNS, the dendritic spine. The principal investigator's previous observations identified three distinct classes of spiny protrusions (filopodia, protospines, and spines) on developing hippocampal dendrites; these protrusions have different dynamic properties (each class progressively more stable), and they emerge sequentially during development. This developmental progression coincides in time with the formation of synapses on dendrites, but the functional relationships between dendritic structure changes, synaptic contact formation, and synaptic activity are poorly understood. The principal investigator will use time-lapse confocal microscopy, electron microscopy, immunocytochemistry, and physiological and molecular perturbations in in vitro rat hippocampal slice and cell cultures to (1) better define the spatiotemporal relationship between synapse formation and dendritic spine development, and (2) determine how cell-cell contacts and synaptic activity may regulate the morphological and molecular development of spines. The principal investigator's primary hypothesis is that nascent synaptic contacts are formed on structurally-dynamic spine precursors, that synaptic activity at these nascent contacts can regulate the developmental plasticity of spine structure, and that this regulation is mediated, in part, by controlling the expression of neural cell adhesion molecules on dendritic surfaces. New information gained from these studies should lead to a fuller understanding of the regulation of synaptic development, and of the relationships between changes in synaptic structure and function. Abnormalities in postsynaptic spine morphology and density have been documented in a variety of developmental disorders and neuropathological conditions such as mental retardation, Alzheimer's Disease, and temporal lobe epilepsy. Moreover, change in synaptic structure and number are thought to underlie normal mental processes, such as learning and memory. Hence, these studies could provide important insight on fundamental mechanisms of synaptic development and plasticity operating over a wide range of normal and abnormal conditions in man.

The long-term goal of this project is to learn how to manipulate axon growth in vivo. There are many instances when this would be an essential part of a therapeutic treatment after injury to the nervous system. In addition, the possible use of transplanted neurons to treat various CNS diseases may depend on these neurons sending axons to appropriate targets. The path growing axons follow during development is regulated by guidance mechanisms consisting of molecular cues that are non- uniformly distributed along the path. A particular role for gradients of attracting and repelling guidance cues has been proposed. A detailed understanding of how gradients of guidance cues direct the growth of axons is necessary to achieve this long-term goal. Recent progress has resulted in the identification and demonstration of in vivo function of 3 non-diffusible gradient molecules involved in the guidance of growth of the Til pioneer axons in the legs of the cockroach embryo. These molecules, localized to the substrate over which the axons grow, include 2 in the basement membrane and l on the surface of the epidermal epithelial cells. The complexity of the interactions between these guidance cues and the growth cone is greater than had been expected. This complexity is expressed in the fact that multiple and functionally redundant cues exist and in that these cues may interact with one another. The cockroach embryo is an ideal system in which to study the complexity of this developmental event. Its anatomical simplicity enables the proximal growth of identified pioneer axons in the leg to be observed in living preparations. In this proposal each of these cues will be molecularly characterized, the details of their ability to guide axon growth both in vivo and in vitro will be examined, as will be the in vivo interactions among them. A fourth molecule distributed in a gradient along the path of axon growth has been identified and experiments are proposed to examine its role in axon guidance. Like many developmental events, axon guidance is also likely to be phylogenetically conserved. The information obtained from studies of this insect are likely to be very relevant to understanding these processes in mammals.

DESCRIPTION (provided by applicant): Microglia are a class of resident brain cells that play key roles in the tissue response to injury or infection of the mammalian brain. The long-term goal of this research program is to understand the cellular and molecular bases of microglial function following brain tissue injury. The present application will use dynamic imaging of cells in live mammalian brain tissues to investigate the molecular basis, regulation, and function of microglial motility. Changes in cellular motility are incorporated into most models of microglial activation following brain injury, but little is known about the motile behaviors of microglia within native brain tissue. Our working hypothesis is that parenchymal microglia represent a heterogeneous population of cells, and that differences in the motile behaviors and intercellular interactions of activated microglial cells are determined by dynamic patterns of cell adhesion molecule expression, providing a basis for functional diversity within the microglial population. Our primary goals here are to elucidate the diversity in cellular response to activation, to define and characterize distinct moti' phenotypes and intercellular interactions of activated microglia, and to determine whether these are regulated by the cytokine, tumor necrosis factor (TNF)-a, acting via the transcription factor, NF-KB. Microglial movements in live rat and mouse brain tissue slices will be analyzed by vital fluorescent staining, 3-D time-lapse confocal imaging, and computer-assisted quantitative image analysis. Retrospective antibody staining following time-lapse observation will enable a quantitative determination of immunophenotypes in relation to the stages of activation and motile phenotypes. Multi-channel time-lapse imaging will be used to characterize functional interactions among microglial cells, and between microglia and dead/dying neurons. Finally, microglial behaviors in slices from TNF receptor knockout mice will be assessed to determine whether TNF regulates motility-based microglial functions. A more complete understanding of the molecular mechanisms of microglial function should yield insight into strategies for regulating the microglial response under a variety of CNS injury conditions. including trauma, epilepsy, stroke, Alzheimer's Disease, and AIDS.

DESCRIPTION (provided by applicant): This application is for a laser scanning confocal imaging system to be devoted to time-lapse or real-time imaging of the dynamic state of live cells in several model systems. The instrument will be integrated into an existing bio-imaging facility that will move into newly renovated space within the Dept. of Biological Sciences. The imaging system will be utilized by a core group of major users and will be available to minor users, both within and outside of the Department, on a limited basis (up to 25% time). The three major users (Drs. Dailey, Lilien, and Soll) will use the instrumentation to accomplish goals related to NIH-funded projects. Dr. Dailey will use time-lapse imaging of neurons in rat brain slices transfected with green fluorescent protein (GFP)-fusion protein constructs to study mechanisms of synapse formation and plasticity. He will test the hypothesis that an adhesion molecule, N-cadherin, regulates synapse formation, plasticity, and maintenance. In another project, Dr. Dailey will characterize the motility behaviors of activated microglia, a type of brain-resident cell that plays a major role in clearing dead cells and cellular debris following brain injury. Simultaneous 2- and 3-channel fluorescence time-lapse imaging will be utilized to study the cell-cell interactions between microglia and other cell types in injured brain tissues. Dr. Lilien will use time-lapse imaging of transfected neurons in developing chick neural retina to determine how adhesion molecules affect the dynamic patterns of axon and dendrite growth. These studies will test hypotheses on the functions of distinct protein domains using perturbational agents that affect specific protein-protein interactions important to N-cadherin function and to cross-talk between N-cadherin and b 1-integrins. Dr. Soll will perform analyses on dynamics of cytoskeletal proteins during cell motility and chemotaxis in live Dictyostelium. The dynamic remodeling of GFP-labeled proteins, including actin, myosins I and II, and the Wiscott-Aldrich protein SCAR, will be analyzed in cells reconstructed every 2 sec, using computer-assisted dynamic image analysis systems (2D and 3D DIAS) developed in the Department's W.M. Keck Image Analysis Facility. The technical expertise of the three primary investigators in time-lapse confocal imaging (Dailey), molecular analysis of adhesion molecules and engineering of cDNAs (Lilien), and motion analysis of cells (Soll) provides a high level of synergism between the projects that will assure their success. Together, these studies will contribute to an understanding of molecular mechanisms of cell growth and directed cell movements related to normal tissue development and to functional remodeling under a variety of pathological conditions in humans.

DESCRIPTION (provided by applicant): Alcohol has well known effects on brain development and function, but its effects specifically on microglial cells (MG) are much less well understood. Here we will utilize in vitro and in vivo mouse brain tissue preparations to study the effects of ethanol on MG activation, motility, migration to injured neurons, and phagocytic clearance of dead cells. We expect that alcohol- induced neuronal injury and apoptotic cell death can be recapitulated in organotypic brain slice cultures derived from neonatal mice, thereby establishing an experimentally tractable in vitro tissue model for studies of glial responses to alcohol-induced neurodegeneration. We hypothesize that alcohol induces brain resident MG to activate rapidly, prior to neuronal cell death, and that MG activation and recruitment to injured neurons via fractalkine signaling promotes neuronal survival and limits secondary neuronal injury. We will test aspects of this hypothesis, first, by assessing the timing of MG activation (changes in MG morphology and gene expression) relative to alcohol-induced initiation of neuronal apoptosis (cleaved caspase-3 labeling) and subsequent cell death (Sytox/Propidium iodide labeling) in mouse hippocampal or cortical slice cultures. To determine whether MG activation precedes, follows, or indeed requires apoptotic neuronal death, we will examine MG activation in slices from mice lacking BAX, an apoptosis regulator protein. Next, we will test whether fractalkine signaling regulates MG activation and recruitment to alcohol-injured neurons in slices from fractalkine receptor null mice (CX3CR1GFP/GFP). Finally, we will use transcranial multiphoton imaging in live, alcohol- treated GFP reporter mice (CX3CR1GFP/+) to examine whether alcohol affects MG basal motility or mobilization to injured neurons in developing or adult cortical brain tissues in vivo. These exploratory studies will yield the first direct observations and analysis of glial cell behaviors in live, intact brain tissues during and following conditions of high blood alcohol and withdrawal. Results from these studies will lay the groundwork for future studies aimed at elucidating the consequences of MG activation for neuroprotection or neurotoxicity. This information will help generate new ideas on the consequences of alcohol for normal brain function as well as for impaired immune surveillance and response to injury in the CNS. PUBLIC HEALTH RELEVANCE: Alcohol-related neuronal injury in humans is an increasing health concern and a growing burden on the economy of our society. Alcohol exposure during critical periods of neuronal development induces damage to or death of neurons, and the response of nearby glial cells, including microglial cells, may promote the survival of injured neurons or exacerbate the injury. The data generated here will help identify responses of microglia to alcohol and alcohol-induced neuronal injury, and thus help guide therapeutic strategies after alcohol-induced brain injury in developing and adult humans.

The Histology and Imaging core supports analysis of mutant ears and connectional defects at all levels in fixed tissue. The goal of this core is to provide the necessary training and certain services to process and image ears at all light and electron microscopic levels, including high resolution multiphoton imaging using multicolor labeling and 3D reconstruction to accelerate analysis of existing and soon to be analyzed mutants or otherwise manipulated ears, including samples of human ears as needed. To this end, the core provides expertise through the core personnel, including a full time RA expertly trained in all techniques needed to evaluate ears and neuronal connections at all stages of development. Reservation of the equipment in this core (three confocal systems, high resolution transmitted and epifluorescence microscopes and dissecting scopes, TEM and SEM, freezing microtomes, vibratomes and ultramicrotomes) will be handled on line. The specific aims of the histology and imaging core are: (1) To provide a range of service (advice and/or assistance) for optimized and standardized histological processing at all levels of light and electron microscopy, including sophisticated multicolor immunocytochemical labeling followed by multiphoton analysis, or combinations of in situ hybridization and immunocytochemistry; (2) To improve data analysis through the availability and use of appropriate image processing and quantitative analysis software; (3) To provide expert training in these techniques to stimulate collaboration between members of the core grant as well as potential additional members currently funded through NIDCD. The histology core closely interacts with other cores such as the molecular core for the generation of in situ probes. The core will be driven by the needs of several researchers to achieve a standardized high resolution analysis of various mouse models generated by genetic or other manipulations. The scientific expertise of the core personnel will enhance the performance of all core related research. An annual workshop that provides overviews of the techniques available in this core and their potential usefulness for research of current and future members of the core will generate a vital interaction toward a hearing research related community at the Univ. of Iowa.