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

本项目的目的是建立一种新的用于分析长期突触可塑性的薄层培养脑片。在前一年的研究中，我们发展了一种长期稳定培养大鼠海马皮质脑片的方法学。今年，我们确定了在切片培养标本中触发突触可塑性的适当刺激条件。除了通过放置在CA3区体细胞层的电极进行常规刺激外，我们还施加了强直刺激(100赫兹1秒)，这使从CA1区体细胞层记录到的群体峰电位幅度增加了两倍多。这种持续20min以上的增加伴随着群体EPSP斜率的增加和峰潜伏期的缩短。这被认为是在新鲜的海马片制备中看到的长期增强的等价物。然而，除了这种典型的反应外，我们还观察到了切片制备…所特有的一些现象更多的包括1)从体层记录到的负EPSP波；2)单一刺激后重复的突触活动。在接下来的研究中，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(并表达APP全蛋白)的培养的海马神经元对施加谷氨酸的反应性显著增强，对去极化的反应性保持不变。因此，提出APP的功能可能是对谷氨酸受体的调制。在上述研究的同时，我们打算用相同的标本来探讨由不活动引起的突触萎缩。河豚毒素阻断自发性突触活动后，神经元出现较少的树突和棘突，提示萎缩的机制可能在该系统中得到了分析。较少

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