HIPPOCAMPAL SYNAPTIC STRUCTURE--PHYSIOLOGY DURING AGING

这是一个关于脑老化的基本膜和突触机制的项目的竞争性延续的提议，该项目已经进行了超过15年。本项目前期研究发现，大鼠海马CAl神经元随着衰老发生了两种主要的电生理变化：1)突触频率增强（促进）功能受损；2)电压门控Ca2+内流增加。这些结果和其他结果都对大脑衰老和痴呆的一般Ca2+假说做出了贡献。在最近的一段时间里，单通道膜片钳配置被用于脑衰老研究，并确定了可用的l型Ca2+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，将测试l型Ca2+通道的增加是衰老哺乳动物神经元功能受损（突触增强或尖峰产生）和神经元死亡易感性的关键机制的具体假设。研究将在大鼠海马切片和长期海马体培养中进行，其中l型Ca2+通道丰富。重复突触激活对成年和老年大鼠海马切片神经元Ca2+瞬态的大小和形貌的影响将被研究，使用快速紫外共聚焦激光扫描显微镜进行Ca2+成像同时与细胞内电生理记录。多种特定通道阻滞剂和激酶调节剂将用于定义关键的Ca2+进入途径。这些研究将确定突触后Ca2+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，并有助于老年大脑神经元的变化。在细胞培养的平行研究中，通过研究具有不同l型通道补体的细胞对兴奋毒性的易感性差异，将测试时间依赖性离子通道变化在细胞死亡中的作用。单通道记录、Ca2+成像、药理学阻断和激酶调节将用于确定坏死和凋亡关键的Ca2+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.