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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS029875-03
Application #
3416799
Study Section
Neurology B Subcommittee 2 (NEUB)
Project Start
1991-08-01
Project End
1994-07-31
Budget Start
1993-08-01
Budget End
1994-07-31
Support Year
3
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Virginia
Department
Type
Schools of Medicine
DUNS #
001910777
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Medrano, S; Steward, O (2001) Differential mRNA localization in astroglial cells in culture. J Comp Neurol 430:56-71
Schauwecker, P E; Ramirez, J J; Steward, O (2000) Genetic dissection of the signals that induce synaptic reorganization. Exp Neurol 161:139-52
Steward, O; Kelley, M S; Schauwecker, P E (1997) Signals that regulate astroglial gene expression: induction of GFAP mRNA following seizures or injury is blocked by protein synthesis inhibitors. Exp Neurol 148:100-9
Steward, O; Trimmer, P A (1997) Genetic influences on cellular reactions to CNS injury: the reactive response of astrocytes in denervated neuropil regions in mice carrying a mutation (Wld(S)) that causes delayed Wallerian degeneration. J Comp Neurol 380:70-81
Schauwecker, P E; Steward, O (1997) Genetic influences on cellular reactions to brain injury: activation of microglia in denervated neuropil in mice carrying a mutation (Wld(S)) that causes delayed Wallerian degeneration. J Comp Neurol 380:82-94
Fujiki, M; Steward, O (1997) High frequency transcranial magnetic stimulation mimics the effects of ECS in upregulating astroglial gene expression in the murine CNS. Brain Res Mol Brain Res 44:301-8
Schauwecker, P E; Steward, O (1997) Genetic determinants of susceptibility to excitotoxic cell death: implications for gene targeting approaches. Proc Natl Acad Sci U S A 94:4103-8
Kelley, M S; Steward, O (1996) The role of postlesion seizures and spreading depression in the upregulation of glial fibrillary acidic protein mRNA after entorhinal cortex lesions. Exp Neurol 139:83-94
Kelley, M S; Steward, O (1996) The process of reinnervation in the dentate gyrus of adult rats: physiological events at the time of the lesion and during the early postlesion period. Exp Neurol 139:73-82
Bonthius, D J; Lothman, E W; Steward, O (1995) The role of extracellular ionic changes in upregulating the mRNA for glial fibrillary acidic protein following spreading depression. Brain Res 674:314-28

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