ANIMAL MODEL AND FUNCTIONAL CHANGES IN AGING

项目2的研究涉及整个计划的三个方面

The research in Project 2 addresses three of the Overall Program Objectives. As a component of Program Objective #1, behavioral studies will be conducted to assess the performance of aged rats on tests of attention, which are sensitive to immunotoxic lesions of cholinergic neurons in the basal forebrain in young rats. This behavioral characterization will provide a background for neurobiological studies in other projects (Projects 1, 5, and 6), and rats tested on these tasks will also be used under Aim #2 in this project to study the response of an immediate early gene to cholinergic activation. For this purpose in situ hybridization histochemistry (ISHH) for c-Fos mRNA will be used to map activation after a low dose of cholinergic agonist (pilocarpine) as a function of and behavioral impairment. This analysis will also address the 3rd Program Objective aimed at the study of signal transduction mechanisms. To achieve that program objective, we will in addition examine protein kinase C (Aim #1). We will assess mRNAs for several isoforms of PKC in hippocampus using ISHH. In a second series of studies, we will examine localization of PKC in cytosol and membrane fractions using Western blotting. These latter experiments will be conducted in two behavioral contexts: after a defined episode of spatial learning and following induction of behaviorally relevant plasticity in the hippocampal formation. The remaining experiments (in Aims 3 & 4) are designed to address specific hypotheses about the role of glucocorticoids in hippocampal aging (Program Objective #4). These studies will initially focus on adrenocorticoid receptor mRNA using solution hybridization assays to determine whether alterations in the aged hippocampus are related to behavioral impairment and to other associated alterations in hippocampal mRNAs, i.e. increased expression of mRNAs for glial fibrillary acidic protein (GFAP) and beta amyloid precursor protein (BAPP). In addition to characterizing the status of these mRNAs in intact young and aged rats, experiments will be conducted to determine whether glucocorticoid exposure plays a necessary and/or sufficient role in the emergence of mRNA signatures of hippocampal aging and hippocampal-depend behavioral impairment. Finally, experiments are designed to examine a possible basis for age-dependent differences in hippocampal glucocorticoid receptor (GR) function. Our studies will focus on AP-1 and its components Jun and Fos, which can determine whether genes regulated by GR are activated or repressed. We will examine the hypothesis that one or more Jun mRNAs are elevated compared to Fos mRNA in aged hippocampus, a condition that could lead to 'switching' GR function from repression to activation. We will then determine whether aging and/or cognitive impairment is associated with increased binding of Jun-related proteins (decreased Fos) to the GFAP-AP1 and/or proliferin composite element using gel shift assays.