Fred J. Roisen - US grants

Gangliosides are membrane-associated glycolipids that have important regulatory roles in many cellular events including differentiation, growth and regeneration. Their concentration is unusually high in neuronal tissue. Active ganglioside synthesis has been found to coincide with periods of intense neuritogenesis and synaptogenesis suggesting that gangliosides play a key role in these processes. We have shown that bovine brain gangliosides mixtures (BBG) enhance axonal sprouting and metabolic activity of primary and established neuronal cell lines. Scanning electron microscopy reveals dramatic changes in surface topography within minutes of ganglioside exposure. The relationship between these surface changes and sprouting is under investigation. An extensive BBG-induced intraneuronal microfilamentous network has been demonstrated with whole-cell electron microscopy. Cytoskeletal disruptive and stabilizing agents are being employed to determine the ultrastructural basis of ganglioside-mediated neuritogenesis. Although eleven different gangliosides increased sprouting of Neuro-2a neuroblastoma, each species elicited morphologically distinct responses. The mechanisms underlying these differences will be examined. The ganglioside GM1 potentiates the action of submaximal levels of Nerve Growth Factor (NGF) on chick sensory ganglia and the adrenal pheochromocytoma PC-12 cells. Liposomal-derived, affinity-purified antibodies directed against the ganglioside GM1 reduce, in a dose-related fashion, NGF-induced axonal sprouting and enzyme induction of these cells. In contrast, antibodies directed against GM2 or GQ1c have almost no effect. These results suggest that membrane-associated ganglioside may function to modulate neurotrophic interactions and have prompted the speculation that the ganglioside GM1 may be specifically involved in the regulation and amplification of NGF. To investigate this hypothesis we will examine the effect of gangliosides and antibodies directed against gangliosides on well defined, in vitro models of neurotrophic interaction. Histochemical and morphological probes, including lectins, toxins, Colcemid and cytochalasin D, will be employed to examine the distribution and fluidity of neuronal membranes as well as the relationship between membrane-associated gangliosides and their cytoskeletal anchorages. These studies will determine the role of gangliosides in neuritogenesis and will provide information on the basic mechanisms of neuronal development and regeneration.

biomedical equipment resource; biomedical equipment purchase;

Recent technical advances have transformed the optical microscope from an information gathering tool into an interactive analytical instrument capable of detecting, manipulating, quantifying and sorting subcellular, cellular and tissue specific properties. This application requests funds for the purchase of an Ultima 312TM Premium Laser Confocal Microscopic workstation (manufactured by Meridian Instruments, Inc.) with enhanced interactive software for data acquisition, storage, and off-line data acquisition. This confocal system was selected because its unique and comprehensive confocal abilities with multiple laser lines and applications software will accommodate the diverse needs of the researchers at the University of Louisville. To maximize user time, the system includes components that permit remote (off-line) data analysis and 3-D reconstruction while the microscope is in use. Among the tasks to be performed are: to obtain highly-purified primary embryonic cell populations; to study Ca2+ metabolism and intracellular pH after electrical and chemical stimulation; to examine co-localization of tRNA biosynthetic enzymes; to perform high resolution in situ hybridization studies; to determine the distribution of membranous components (i.e. receptors, complex carbohydrates) and their relationships to cytoskeletal organelles during a variety of dynamic cellular phenomenon including phagocytosis and neuritogenesis; and to obtain 3-D reconstructions for cytoarchitectural and developmental morphometric comparisons. The 16 research paradigms of the 32 faculty members in this application are not inclusive of all projects that would utilize the Ultima, but are intended to demonstrate the breadth of our needs. The instrument will be housed in the Core Analytical Microscopy Laboratory (CAML) which has served as the sole microscopy laboratory for all campuses of the University for the past four years. Being the first confocal microscopy system in the CAML, it would enhance significantly our optical microscopic capabilities, thereby providing for cuttingedge technology for research and the training of CAML users, graduate students and postdoctoral fellows. The research and outreach mission of our "Developmental and Molecular Neurobiology Program" funded by an NSF KY/EPSCoR contract will be greatly facilitated by this instrumentation. Furthermore, recent faculty recruitment in the biological sciences has been hampered by the lack of a confocal microscope. Therefore, in addition to facilitating research and training of investigators and students at U of L, the acquisition of the Ultima should assist future faculty recruitment in several departments and thus facilitate the strategic plan of the University to become a research institution. The genuine widespread need for this equipment is mirrored by the fact that more than 50% of the funds needed to purchase the confocal scanning laser microscopic workstation have been raised from the office of the University Vice President for Research, Dean of the Medical School, and 10 different departments in several schools as well as individual users.

stem cell transplantation; respiratory epithelium; nervous system regeneration; central nervous system;

microscopy; nervous system regeneration; spinal cord imaging /visualization; spinal cord injury; bioimaging /biomedical imaging;

bioimaging /biomedical imaging; spinal cord imaging /visualization

respiratory epithelium