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

本项目的目的是建立薄层培养脑切片作为长期突触可塑性分析的新系统。在去年的研究中，我们开发了一种长期稳定培养大鼠海马皮质切片的方法。今年，我们确定了触发切片培养样本中突触可塑性的适当刺激条件。除了通过放置在 CA3 区域体细胞层的电极进行常规刺激外，我们还应用了强直刺激（100Hz 1 秒），这使得从 CA1 区域体细胞层记录的群体尖峰幅度增加了两倍以上。这种增加持续了 20 分钟以上，伴随着群体 EPSP 斜率的增加和尖峰潜伏期的缩短。这被认为相当于新鲜海马切片制备中所见的长期增强作用。然而，除了这种典型的反应之外，我们还观察到了切片培养物制备所特有的一些现象，其中包括 1) 从体细胞层记录的负 EPSP 波； 2）单一刺激后的重复突触活动。在后续的研究中应该有必要抑制（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，我们尝试了对培养的神经元进行活体染色。为了规避亲脂性染料的毒性，我们打算借助腺病毒载体引入水母绿色荧光蛋白（GFP）的cDNA。在此之前，作为练习，我们介绍了淀粉样前体蛋白（APP）的 cDNA，其功能目前尚不清楚。接受腺病毒（并因此表达APP全蛋白）的培养海马神经元对所应用的谷氨酸表现出显着更高的反应性，而对去极化的反应性未改变。因此提出了APP的功能是调节谷氨酸受体的可能性。 与上述研究并行，我们打算使用相同的样本来探讨不活动引起的突触萎缩。通过用河豚毒素阻断自发突触活动，神经元显示出较少数量的树突分支和棘，表明可以在该系统中分析萎缩的机制。较少的

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