Astrocytes play essential roles in brain homeostasis and in the brain's response to injury. With freeze fracture techniques, we have identified a membrane-associated protein which is concentrated in astrocytic processes investing blood vessels and forming the surface of the brain. Recent evidence indicates that this protein, which we have termed """"""""assemblies"""""""" because of its appearance in freeze fractured preparations, is of central importance in the maintenance of brain extracellular fluid ionic composition. The structure of assemblies is liable in the setting of hypoxic brain injury. Assemblies may be directly involved in the pathogenesis of astrocytic swelling, a component of cerebral edema in a variety of clinical disorders. Two independent, but complementary strategies have been devised to determine the nature of this protein, its role in normal brain function, and its role in the brain's response to injury. First, freeze fracture techniques are being used to study factors influencing the differentiation of this aspect of membrane structure in cultured astrocytes, with particular attention to interactions between astrocytes and endothelial cells. The response of astrocytes to in vitro conditions mimicking brain ischemic injury will be examined with several methods, including rapid freezing and freeze fracture. Second, monoclonal antibodies are being generated which bind selectively to astrocytic membranes. The principal intent is to identify antibodies which bind to assemblies, but other monoclonal antibodies generated in the process are powerful tools for the study of astrocyte development and differentiation. It has been possible, using treatment of cultured astrocytes with forskolin or with dexamethasone, to identify bands in electrophoretic gels which correlate with the presence of assemblies. Such bands will be used as immunogen, as well as plasmalemma fractions from cultured astrocytes. Once the specificity of the monoclonal antibodies has been demonstrating by showing binding to assemblies in freeze fractured preparations, the antibodies can be used to develop quantitative assays for the protein represented by assemblies, and to identify its function. As more is learned about this property of astrocytes, it may be possible to design better responses to the clinical problems of astrocytic swelling in ischemic and traumatic injury.
