In the adult brain recurrent seizures induce distinct patterns of change in levels of mRNAs for members of the nerve growth factor (NGF) family of neurotrophins (NGF, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 [NT-3]); these changes precede differential alterations in levels of mRNAs for various neuropeptides and at least one subunit of the non-NMDA glutamate receptor. These findings suggest that physiological activity modulates neurotrophin expression and, thereby, regulates the biosynthetic activities of trophin-responsive cells. The proposed projects address the first aspect of this hypothesis and, in particular, are designed to more funny characterize the dynamic properties of neurotrophin mRNA expression and to test predictions as to cellular mechanisms involved. There are five specific aims. (1) Through analysis of the colocalization of the neurotrophin mRNAs with each other and with glutamic acid decarboxylase mRNA it will be determined (la) if the neurotrophin mRNAs are colocalized and differentially regulated by activity in single cells and (lb) if neurotrophin expression by GABAergic neurons is unaffected by seizures. (2) Run-on assay and in situ hybridization to mRNA intron sequences will be used to determine if seizure-induced changes in neurotrophin mRNA content involve changes in mRNA synthesis. (3) Controlled stimulation of afferents to hippocampus and olfactory cortex will be used to determine the threshold and time courses of changes in activity-driven neurotrophin expression and if there are regional differences in the parameters of the neurotrophin mRNA response. This will include a test of the hypothesis that subseizure physiological activity regulates neurotrophin mRNA expression. (4) In vitro hippocampal explants will be used to determine whether depolarization-induced increases in NGF and BDNF mRNA are blocked by protein synthesis inhibition; this will test the hypothesis that increases in BDNF and NGF mRNA contents are immediate-early gene responses. (5) The last studies will explore the hypothesis that late changes in NGF mRNA expression after seizures are trophically induced. Exp. 5a will determine if seizures increase levels of mRNAs for acidic fibroblast growth factor (aFGF), basic FGF and interleukin-1beta prior to late increases in NGF mRNA content and Exp. 5b will determine if these cytokines increase the NGF mRNA content of hippocampal neurons in explant culture. Quantitative in situ hybridization and S1 nuclease protection assays will be used to quantify MRNA content. These studies should elucidate mechanisms that regulate the expression of brain neurotrophins thought to be critical for the survival of diverse populations of central neurons. Moreover, the results will indicate if activity dependent regulation of neurotrophin expression is an ongoing property of central neurons and, therefore, a likely mechanism through which activity influences the structure, function, and viability of the adult brain.
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