HIPPOCAMPAL SYNAPTIC STRUCTURE--PHYSIOLOGY DURING AGING

这是一个关于大脑老化的基础膜和突触机制的项目的竞争性延续的提案，该项目已经持续了15年以上。在过去的研究中发现，随着年龄的增长，大鼠海马神经元发生了两个主要的电生理变化：1)突触频率增强(易化)受损；2)电压门控钙内流增加。这些结果和其他研究结果共同构成了关于大脑老化和痴呆的普遍的钙离子假说。最近，单通道膜片钳结构被用于脑老化的研究，并发现可用L型钙通道膜密度增加是在细胞水平上看到的随年龄变化的潜在分子基础。在这里，L类钙通道的增加是衰老哺乳动物神经元功能受损(突触增强或棘波产生)和神经元易死亡的关键机制的具体假设将得到检验。研究将在大鼠海马片和长期培养的海马区进行，其中L型钙通道丰富。本研究将在细胞内电生理记录的同时，用快速紫外兼容激光共聚焦扫描显微镜研究重复突触激活对成年和老年大鼠海马片神经元内钙瞬变的幅度和地形图的影响。多种特定的通道阻滞剂和激酶调节剂将被用来定义关键的钙离子进入途径。这些研究将确定突触后钙瞬变，特别是通过L通道，是否可以调节神经元的短期突触可塑性，并有助于老年脑神经元的变化。在细胞培养的平行研究中，将通过研究具有不同L类型通道补体的细胞对兴奋性毒性易感性的差异，来测试依赖于时间的离子通道变化在细胞死亡中的作用。单通道记录、钙成像、药物阻断和激酶调节将被用来确定对坏死和凋亡至关重要的钙来源。

This is a proposal for a competitive continuation of a project on basic membrane and synaptic mechanisms of brain aging that has been ongoing for over 15 years. The past periods in this project have found two main electrophysiological alterations in rat hippocampal CAl neurons with aging: 1) impaired synaptic frequency potentiation (facilitation); and 2) an increase in voltage-gated Ca2+ influx. These results and others have contributed to the general Ca2+ hypothesis of brain aging and dementia. In the most recent period, the single channel patch clamp configuration was adapted for brain aging studies and identified an increase in the membrane density of available L-type Ca2+ channels as a potential molecular basis for the changes with aging seen at the cellular level. Here, the specific hypothesis that the increase in L-type Ca2+ channels is a key mechanism in both impaired function (synaptic potentiation or spike generation) and neuronal vulnerability to death of aged mammalian neurons will be tested. Studies will be conducted in rat hippocampal slices and long-term hippocampal cultures in which L-type Ca2+ channels are enriched. Effects of repetitive synaptic activation on the magnitude and topography of Ca2+ transients in hippocampal slice neurons of adult and aged rats will be studied, using a rapid UV-compatible confocal laser scanning microscope for Ca2+ imaging simultaneously with intracellular electrophysiological recording. Multiple specific channel blockers and kinase modulators will be used to define critical Ca2+ entry pathways. These studies will determine whether postsynaptic Ca2+ transients, particularly through L-channels, can modulate neuronal short-term synaptic plasticity and contribute to changes in aged brain neurons. In parallel studies of cell cultures, the role of time-dependent ion channel changes in cell death will be tested, by investigating differences in vulnerability to excitotoxicity in cells with different complements of L-type channels. Single channel recording, Ca2+ imaging, pharmacologic blockade and kinase modulation will be used to define Ca2+ sources critical for necrosis and apoptosis.