This project seeks to identify the inter-cellular signals that up-regulate astrocyte gene expression following CNS injury, and characterize their mode of action. Studies to date have revealed that the levels of the mRNA for the astrocyte-specific intermediate filament protein GFAP are dramatically up-regulated following CNS lesions. Preliminary evidence suggests that the up-regulation occurs in response to three types of signals: 1) injury-induced bursts of neuronal activity; 2) a diffusible agent present in the CSF of brain-injured animals; and 3) proximity of degeneration debris. Our experiments seek to characterize the mode of action of these different intracellular signals, define their relative contribution to the increases in GFAP expression after brain injury, and develop ways to modify the reactive changes in astroglia by directly manipulating their gene expression. To define the contribution of lesion-induced bursts of neuronal activity, we will: A) produce electrolytic brain lesions while neuronal activity is blocked with TTX, and B) Test the hypothesis that decreases in neuronal activity affect GFAP mRNA levels by blocking activity with TTX ; C) Elicit intense electrical activity without producing lesions using chronic neurophysiological techniques. To define the role of diffusible factors released into the ventricular system, we will harvest CSF from brain-injured animals, and evaluate whether this CSF up-regulates glial gene expression in the hippocampus when injected into the ventricles of control animals. The contribution of degeneration debris will be revealed by the spatial and temporal pattern of altered gene expression, and by elimination when the contributions of the other factors are known. Immunocytochemical and biochemical techniques will be used to define the relationship between the time course of changes in GFAP mRNA levels and GFA protein levels. We will also attempt to identify the inter-cellular signals that are responsible for activity-induced upregulation of GFAP mRNA levels. Two classes of inter-cellular signals seem most likely to be involved: A) neurotransmitters; and B) extracellular ions, particularly K+. The potential role of these signals will be assessed using pharmacological blocking agents in vivo, and by evaluating their effect on astrocyte gene expression in vitro. To evaluate the role that glial reactivity plays in regulating neuronal growth following injury, we will determine whether the time course or extent of postlesion neuronal growth is affected by manipulations with after the time course and spatial pattern of increases in GFAP mRNA levels following lesions. These experiments will allow us to test hypotheses concerning the role of glial reactivity in postlesion neuronal plasticity, which in turn may lead to the development of manipulations which can promote regenerative growth of neurons after injury.
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