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Toward visualization of memory ; Analysis of synaptic plasticity utilizing a novel model system

走向记忆的可视化；

基本信息

批准号：
07458213
负责人：
OGURA Akihiko
金额：
$ 4.42万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

The aim of this project is to establish the thin-layr-cultured brain slice as a novel system for analysis of long-term synaptic plasticity.In the research of the previous year, we developed a methodology for the long-term stable culture of rat hippocampal cortical slice. This year we determined appropriate stimulus conditions for triggering the synaptic plasticity in the slice culture specimen. In addition to regular stimuli through an electrode placed in the somatic layr of CA3 area, we applied a tetanic stimuli (100Hz 1sec), which produced a more-than-two-fold increase in population spike amplitude recorded from the somatic layr of CA1 area. This increase, lasting for more than 20 min, was accompanied by an increase of population EPSP slope and a shortening of spike latency. This is concluded as the equivant of long-term potentiation seen in fresh hippocampal slice preparation. Besides such typical response, however, we observed some phenomena peculiar to the sliceculure prepartion, … More which include 1) negative EPSP waves recorded from the somatic layr ; 2) repetitive synaptic activity following a single stimulus. It should be necessary to suppress (or make use of) these anomalous synaptic behavior in the following researches.For morphological examinations on the brain slice-culture specimens, we tried live staining of cultured neurons. To circumvent the toxicity of lipophilic dyes, we intended to introduce a cDNA of jellyfish green fluorescent protein (GFP) by the aid of adenovirus vector. Before that, as an exercise, we introduced a cDNA of amyloid precursor protein (APP), whose function is so far unclear. The cultred hippocampal neurons which received adenovirus (and thus expressing APP holoprotein) showed significantly higher responsiveness to applied glutamate, leaving the responsiveness to depolarization unaltered. The possibility is thus raised that the function of APP is the modulation of glutamate receptors.In parallel with above researches, we intended to approach to the inactivity-caused atrophy of synapses using the same specimen. By the blockage of spontaneous synaptic activity with tetrododoxin, neurons showed lesser number of dendritic branching and spines, indicating that the mechanism of atrophy might be analyzed in this system. Less

本课题的目的是建立一种新的脑片薄层培养系统，用于突触可塑性的长期研究。在前一年的研究中，我们建立了一种长期稳定培养大鼠海马脑片的方法学。今年，我们确定了适当的刺激条件，触发突触可塑性的切片培养标本。除常规刺激外，还加用强直刺激（100 Hz/sec），使CA_1区皮层记录到的群体锋电位幅度增加2倍以上。这种增加持续20 min以上，并伴随着群体EPSP斜率的增加和锋电位潜伏期的缩短。这被认为是在新鲜海马切片制备中观察到的长时程增强的等价物。然而，除了这种典型的反应外，我们还观察到一些切片培养所特有的现象， ...更多信息其中包括1）从体细胞层记录到的负性EPSP波; 2）单一刺激后的重复性突触活动。在以后的研究中有必要抑制（或利用）这些异常的突触行为。为了对脑切片培养标本进行形态学检查，我们尝试了培养神经元的活染色。为了克服亲脂性染料的毒性，我们打算通过腺病毒载体将水母绿色荧光蛋白（GFP）的cDNA导入细胞。在此之前，作为练习，我们引入了淀粉样前体蛋白（APP）的cDNA，其功能迄今尚不清楚。接受腺病毒（从而表达APP全蛋白）的培养海马神经元对谷氨酸的反应性显着更高，对去极化的反应性不变。与此同时，我们还用同一标本探讨了突触失活引起的突触萎缩。通过用tetrododoxin阻断自发性突触活动，神经元显示出较少的树突分支和棘，表明可以在该系统中分析萎缩的机制。少