ANIMAL MODEL AND FUNCTIONAL CHANGES IN AGING

项目2中的研究涉及总体计划中的三个 目标。作为计划目标1的一个组成部分，行为研究 将进行评估老化大鼠在测试 注意，对胆碱能免疫毒性损害敏感的 幼年大鼠基底前脑的神经元。这一行为 特征化将为神经生物学研究提供背景 其他项目(项目1、5和6)，以及在这些任务上测试的RAT将 也在本项目的目标2下使用，以研究 即刻早期基因对胆碱能的激活作用。为此目的，就地 将使用c-Fos mRNA的杂交组织化学(ISHH)来定位 小剂量胆碱能激动剂(匹罗卡品)作为 功能和行为障碍。这一分析还将解决 以信号转导研究为目标的第三个计划目标 机械装置。为了实现这一计划目标，我们还将 检查蛋白激酶C(目标1)。我们将评估以下几个方面的mRNA 用ISHH法研究海马区PKC亚型。在第二系列研究中， 我们将研究PKC在胞浆和膜组分中的定位 使用Western blotting。后两个实验将分两个阶段进行 行为背景：在一段明确的空间学习和 在诱导海马区行为相关可塑性之后 队形。其余的实验(在目标3和4中)旨在 解决有关糖皮质激素在脑内的作用的具体假设 海马区老化(方案目标4)。这些研究将初步 用溶液杂交方法研究肾上腺皮质激素受体基因的表达 以确定老年海马区的改变是否与 行为障碍和其他相关的海马区改变 MRNAs，即胶质纤维酸性的mRNAs表达增加 蛋白质(GFAP)和β淀粉样前体蛋白(BAPP)。除了……之外 描述这些mRNAs在完整的年轻和老年大鼠中的状态， 将进行实验以确定是否接触糖皮质激素 在信使核糖核酸的产生中起到必要的和/或充分的作用 海马区老化和海马区依赖行为的特征 减损。最后，设计实验来检验可能的基础 海马糖皮质激素受体(GR)的年龄相关性差异 功能。我们的研究将集中在AP-1及其组分Jun和Fos， 它可以确定受GR调控的基因是被激活还是 被压抑。我们将检验这样的假设：一个或多个Jun mRNAs是 与老年海马区Fos mRNA相比，这种情况可能 导致将GR功能从抑制转换为激活。我们会 然后确定衰老和/或认知障碍是否相关 随着Jun相关蛋白结合增加(Fos减少) 凝胶移位分析GFAP-AP1和/或增殖蛋白复合元件。

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.