The studies proposed in this application focus on three related issues of fundamental importance in understanding the structure and function of neuronal and astrocytic membranes in the mammalian central nervous system: 1) structure, composition, and function at synaptic junctions; 2) cytoskeletal and membrane structure in axonal and dendritic growth cones; and 3) composition and function of specializations of astrocyte membrane structure. We have found, using freeze fracture and thin-section study of rapidly frozen tissue, that Purkinje cell dendritic spines in adult cerebellar cortex have a complex cytoskeletal structure which is in a position to constrain the shape of a spine. This cytoskeletal system might be able to effect changes in the shape of the spine, which in turn would produce significant changes in synaptic function; such a mechanism might have a role in learning and memory. We plan to determine whether this cytoskeleton is a general feature of spines, and whether changes in cytoskeletal structure and spine shape accompany long-term synaptic potentiation and denervation. There is also a system of filaments interlacing among synaptic vesicles in axonal cytoplasm which may transport synaptic vesicles to the active zone. Immunocytochemical techniques will be used to determine the composition of these synapse-related filament systems and of cytoskeletal structures in growth cones. We will apply rapid freezing techniques to examine the manner in which membrane is added during axonal and dendritic growth in vivo and in vitro. We have described a specialization of membrane structure in astrocytes which is concentrated at the blood-brain barrier and opposite the cerebrospinal fluid compartment, and have found that swelling of astrocytic processes during ischemic injury is associated with dissolution of this membrane protein. Astrocytes express this membrane specialization in primary cultures, and we will examine its response to glucocorticoids (used in the treatment of cerebral edema), high concentrations of potassium, and co-cultivation with blood vessels and neurons.
