Synaptic substrates of age-dependent memory deficits

 描述(申请人提供)：虽然衰老本身是阿尔茨海默病(AD)的最大风险因素，但许多有记忆问题的老年人最终并不患有AD。在这些人中，尽管有重叠的认知症状，但缺乏值得诊断为AD的病理。这项研究计划的主要目标是在大鼠中模拟这种与年龄相关的认知异质性，然后使用高分辨率技术来揭示与年龄相关的记忆形式下降的突触和树突底物，这需要海马体。作为海马区的主要输出，CA1区一直是我们等人研究的主要目标。事实上，过去的研究表明，虽然CA1中没有主要的与年龄相关的突触和神经元丢失，但更微妙的与年龄相关的变化串通在一起，改变了神经元的活动模式和行为可塑性。重要的是，这种变化只存在于老年大鼠的一小部分中，而其他相同年龄段的大鼠在Morris水迷宫(MWM)学习和痕迹眨眼条件反射(TEBC)中显示出年轻成人样的习得。因此，老年大鼠的这种认知异质性提供了一个重要的机会，既揭示了与年龄相关的记忆衰退的底物，也揭示了记忆保存的底物。在这项更新申请中提出的实验将继续揭示与年龄相关的认知异质性的突触和树突基础，但通过建立在我们过去的发现和概念和技术复杂性的基础上做到这一点。具体地说，在目标1中，我们将用MWM学习和TEBC来描述年轻的成年和老年的F344xBN F1杂交大鼠的行为特征，然后用双光子激光扫描(2PLSM)钙成像和双光子谷氨酸(2PGlu)渗透到单个基础、倾斜和簇状树突的离散微域的棘上来探测突触钙信号。在这一高分辨率功能探测之后，AIMS 2和AIMS 3将使用免疫金场发射扫描电子显微镜(IFESEM)和免疫荧光阵列断层扫描(IAT)从形态上重建成像的树突，然后从蛋白质组学上重建树突的信号网络。目标2将专注于参与突触和树突电压信号和整合的离子通道的相互作用组。目标3将探测成像的树突，寻找参与调控突触和树突钙信号的交互作用组的离子通道。利用这些AIMS提供的信息，AIM 4将通过病毒转染法挽救与年龄相关的记忆障碍和信号异常，以上调或下调与认知障碍或保存相关的离子通道的表达。这样的“拯救”将通过行为测试、2PLSM钙成像、2P谷氨酸去除以及iFESEM和IAT重建蛋白质组显微镜来探索。总之，在拟议的AIMS中的实验将以分子精确的方式识别老年大鼠树突状细胞信号和记忆功能的关键调节因子，揭示“成功的”和“不成功的”认知衰老，并提供关于可能减缓或防止与衰老相关的认知衰退的可行药物靶点的关键信息。

 DESCRIPTION (provided by applicant): Though aging itself is the biggest risk factor for Alzheimer's disease (AD), many aged persons with memory problems do not ultimately suffer from AD. In such persons, pathology that would merit AD diagnosis is absent, despite the overlapping cognitive symptomatology. The principal goal of this research program is to model such "normal" age-related cognitive heterogeneity in rats and then use high-resolution techniques to reveal the synaptic and dendritic substrates of age-associated decline in forms of memory that require the hippocampus. As the primary output of the hippocampus, region CA1 has been a primary target of such investigation by us and others. Indeed, past studies have revealed that while major age-related synapse and neuron loss are absent in CA1, more subtle aging-linked changes collude to modify neuronal activity patterns and behavioral plasticity. Importantly, such alterations are present in only a subset of aged rats, with others the same chronological age showing young adult-like acquisition on Morris Water Maze (MWM) learning and trace eyeblink conditioning (TEBC). Such cognitive heterogeneity among aged rats, then, provides an important opportunity to reveal both the substrates of age-related memory decline as well as the substrates of memory preservation. The experiments proposed in this renewal application will continue to reveal the synaptic and dendritic substrates of age-related cognitive heterogeneity, but do so by building on our past findings with conceptual and technical complexity. Specifically, in Aim 1, we will behaviorally characterize young adult and aged F344xBN F1 hybrid rats with MWM learning and TEBC and then probe synaptic Ca2+ signaling with 2-photon laser scanning (2PLSM) Ca2+ imaging and 2-photon glutamate (2Pglutamate) uncaging onto spines in discrete microdomains of individual basal, oblique, and tuft dendrites. Following this high-resolution functional probing, Aims 2 and 3 will use immunogold field emission scanning electron microscopy for backscattered electrons (iFESEM) and immunofluorescence array tomography (iAT) to morphologically reconstruct the imaged dendrites and then proteomically reconstruct the dendrites' signaling networks. Aim 2 will focus on an interactome of ion channels involved in synaptic and dendritic voltage signaling and integration. Aim 3 will probe the imaged dendrites for ion channels involved in an interactome that governs synaptic and dendritic Ca2+ signaling. Using the information provided by these Aims, Aim 4 will rescue age-related memory impairments and signaling abnormalities with viral transfection to up or down-regulate expression of ion channels linked to cognitive impairment or preservation. Such "rescue" will be probed with behavioral testing, 2PLSM Ca2+ imaging, 2Pglutamate uncaging, and reconstructive proteomic microscopy with iFESEM and iAT. Together, the experiments in the proposed Aims will identify the key regulators of dendritic signaling and memory function in aged rats with molecular precision, reveal deep insight into both "successful" and "unsuccessful" cognitive aging, and provide critical information about viable drug targets that could slow or prevent aging-linked cognitive decline.