The goal of this project is to analyze the physiological roles of gap junctions formed by astrocytes. The overriding hypothesis is that gap junctions are important in critical astrocytic functions and that disruption of coupling will impact adversely on these functions. We will apply standard methods of modulation of gap junctional communication, all at least partially developed in this Program, and new methods including gene knockout by homologous recombination and antisense strategies. An important further approach will be to restore coupling by transient transfection or viral transduction with wild type Cx43, with mutant Cx43 or with another connexin that differs functionally from Cx43.
Specific Aim 1. Characterize the connexin(s) expressed by astrocytes. The principal connexin in astrocytes is Cx43; we have strong evidence that Cx40 is also expressed, although to a lesser degree. Astrocytes from a Cx43 knockout mouse exhibit infrequent weak electrical and dye coupling; the junctional channel properties are distinct from those of Cx43.
Specific Aim 2. Test the hypothesis that gap junctional coupling between astrocytes mediates propagation of Ca2+ waves and facilitates secretion of neurotrophic substances and uptake of neurotransmitters. Blockers of gap junctional communication that depend on connexin nucleotide or protein sequences will allow highly specific block of junctional coupling. Astrocytes from the Cx43 knockout mouse also provide a preparation with reduced coupling.
Specific Aim 3. Test in culture the hypothesis that factors secreted by neurons and other cell types modulate levels, distribution and properties of astrocyte gap junctions. The incidence of gap junctions between astrocytes changes following trauma in vivo and coupling can be modulated by neurotransmitters in vitro. We will attempt to reproduce in vitro the changes in astrocyte connexins that occur in reactive gliosis.
Specific Aim 4. Characterize the changes in coupling of astrocytes in reactive gliosis. We will determine whether there are alterations in coupling associated with the changes in Cx43 distribution and immunoreactivity. We will determine whether other changes in gliosis, such as up- regulation of GFAP, are affected by modifying coupling.
Specific Aim 5. Test the hypothesis that gap junctions between astrocytes decrease susceptibility to epileptogenic stimuli. We will use brain slices in culture and determine effects on seizure thresholds of modifying junctional coupling. This broad based study should greatly increase our understanding of the roles of astrocytes and provide possibilities of therapeutic strategies for neural protection against trauma, for prevention of neurodegeneration and for amelioration of disease processes.
