HIPPOCAMPAL SYNAPTIC STRUCTURE--PHYSIOLOGY DURING AGING

这是一个关于大脑衰老的基本膜和突触机制的项目的竞争性继续的建议，该项目已经进行了15年以上。在该项目的过去阶段已经发现大鼠海马CA 1神经元中随着衰老的两个主要电生理学改变：1）受损的突触频率增强（易化）;和2）电压门控Ca 2+内流的增加。这些结果和其他结果有助于脑老化和痴呆的一般Ca 2+假说。在最近的时期，单通道膜片钳配置适用于脑老化研究，并确定了可用的L-型Ca 2+通道的膜密度的增加作为在细胞水平上观察到的衰老变化的潜在分子基础。在这里，具体的假设，即增加L-型Ca 2+通道是一个关键机制，在受损的功能（突触增强或尖峰生成）和神经元的脆弱性，老年哺乳动物神经元死亡将进行测试。研究将在大鼠海马切片和长期海马培养物中进行，其中L型Ca 2+通道富集。重复突触激活对成年和老年大鼠海马脑片神经元中Ca 2+瞬变的幅度和地形的影响将被研究，使用快速UV兼容的共聚焦激光扫描显微镜进行Ca 2+成像，同时进行细胞内电生理记录。多种特异性通道阻滞剂和激酶调节剂将用于确定关键的Ca 2+进入途径。这些研究将确定突触后Ca 2+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。在细胞培养物的平行研究中，将通过研究具有不同L型通道补充物的细胞中对兴奋性毒性的脆弱性差异来测试时间依赖性离子通道变化在细胞死亡中的作用。将使用单通道记录、Ca 2+成像、药理学阻断和激酶调节来确定坏死和凋亡的关键Ca 2+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.