Studies of Excitatory Amino Acid Receptors in the Hippocampus-from a viewpoint of neurotransmitter interactions-

目前的研究是表征大脑中的兴奋性氨基酸受体，重点是海马和小脑。所采用的方法有结合实验、体外受体放射自显影和受体蛋白化学分析。重点研究了NMDA受体复合物各亚组分结合特性和分布的年龄相关性变化。NMDA受体正在形成一个由谷氨酸结合位点、甘氨酸结合位点、多胺结合位点、离子通道等众多亚组分组成的结构复杂的复合物。我们发现Triton X-100预处理后，NMDA识别位点的特异性结合明显增强。该方法使阐明NMDA受体复合物的结合特性成为可能。通过甘氨酸、d -丝氨酸、6-7-二氯喹啉- 2,3 -二酮（DCQX）、7-氯代喹啉酸（7-CIKYNA）对NMDA结合的抑制实验，分析了其抑制机制。甘氨酸有效抑制NMDA结合已经是一个公认的事实。我们进行了7-chlorokynurena （7-CIKYNA）对甘氨酸结合位点的识别实验，指出该物质可用于标记士的宁不敏感的甘氨酸结合位点。关于多胺结合位点，我们在海马中进行了h -亚精胺3结合实验，发现多胺增强了H-MK 801的结合。进一步的实验是关于二价阳离子如何。对抑制实验的影响。研究还扩展到海马和小脑之间NMDA受体结合特性的比较。受体蛋白化学部分，对nmda敏感的谷氨酸结合蛋白进行溶解和部分纯化。纯化后的蛋白具有良好的结合能力。采用体外放射定量自显影技术研究大鼠脑兴奋性氨基酸受体的年龄相关性变化。l-谷氨酸受体可分为三种亚型，分别为n -甲基- d -天冬氨酸受体（NMDA）、半胱氨酸受体和盐酸盐受体。其中，NMDA亚型在学习记忆和神经元呼叫死亡中尤为重要。在这项研究中，我们。专注于NMDA受体复合物实验选用2月龄（青壮年）和21月龄（大月龄）雄性Fischer 344品系大鼠。NMDA受体在海马CAl区密度最高，尤其是在辐射层和取向层。邻近的金字塔层的结合浓度相对较低。大脑皮层也含有高浓度的NMDA受体，并表现出区域和层状变化。在尾核-壳核、嗅球、伏隔核、中隔、丘脑和杏仁核中发现了中等水平的受体。中脑区域和小脑皮层显示出低水平的受体。在NMDA受体复合物中，以h -甘氨酸标记的士的宁不敏感甘氨酸受体在老年大鼠脑前脑区严重减少。在中脑区，h -甘氨酸结合3s蛋白随年龄的平行下降幅度小于端脑区。铈中ii-甘氨酸结合位点没有明显变化。另一方面，H-CPP （NMDA识别位点的二核苷酸）的结合位点在前脑区域略有下降。老龄大鼠海马H-CPP结合位点无明显变化。我们的研究结果表明，在衰老过程中，甘氨酸识别位点对NMDA受体的调节异常在神经元变性中起重要作用。少

The present study is to characterize excitatory amino acid receptors in the brain, focusing on the hippocampus and cerebellum. The used methods were binding experiments, in vitro receptor autoradiography and chemical analysis of receptor proteins. Emphasis was also put on age-related changes of binding characters and distributions of each subcomponent of NMDA receptor complex.NMDA receptor is forming a complex with complicated structure consisting of numerous subcomponents such as glutamate binding site, glycine binding site, polyamine binding site and ion channel.We found out that specific binding of NMDA recognition site was markedly enhanced' by pretreatment of Triton X-100. . This method made it possible to elucidate binding characteristics of NMDA receptor complex. More concretely, inhibition experiments of NMDA binding were done by glycine, D-serine, 6-7-di-chloroquinoxaline-2, 3-dione (DCQX), 7-chlorokynurenate (7-CIKYNA) and mechanisms of these inhibitions were analysed.It has … More been an established fact that glycine potent iates NMDA binding. We performed experiments on recognition of glycine binding sites by 7-chlorokynurena (7-CIKYNA) and pointed out that this substance is useful for labelling strychnine-insensitive glycine binding sites. In relation with polyamine binding site, we performed H-spermidine 3binding experimens in the hippocampus and noticed that polyamine potentiated H-MK 801 binding.Further experiments were done on how bivalent cations. influence on these inhibition experiments. Studies were also extended to comparison of binding characteristics of NMDA receptor between the hippocampus and cerebellum.As for the part of receptor protein chemistry, NMDA-sensitive Hglutamate binding protein was solubilized and partially purified. The purified protein showed excellent binding ability.Age-related changes of excitatory amino acid receptors were investigaed in the rat brain through quantitative in vitro autoradiography. Receptors for L-glutamate have been classified into three subtypes, termed the N-methyl-D-aspartate (NMDA), quisqualate and kainate types. Among them, it is demonstrated that the NMDA subtype is particularly important in learning and memory and in neuronal call death. In this study, we. focused on the NMDA receptor complex. Fischer 344 strain male rats aged 2 (young adult) and 21 (old) months were used in these experiments. The NMDA receptors showed the highest density in the CAl region of the hippocampus, especially in the stratum radiatum and stratum oriens. The adjacent stratum pyramidale had a relatively low concentration of binding. The cerebral cortex also contained high concentrations of the NMDA receptors and exhibited both regional and laminar variations. Moderate levels of receptors were found in the caudate-putamen, olfactory bulb, nucleus accumbens, septum, thalamus and amygdala. Midbrain regions and the cerebellar cortex showed low levels of receptors. Among the NMDA receptor complex, strychnine-insensitive glycine receptors which were labeled with H-glycine were severely reduced in tylencephalic regions in the aged rat brain. In midbrain regions, decreases of 'H-glycine binding 3s ites in parallel with age remained less than those in the telencephalon. ii-glycine binding sites in the cereyellum were not significantly changed. On the -other hand, binding sites of H-CPP (an anta 2onist for an NMDA recognition site) declined slightly in the forebrain area. H-CPP binging sites in the hippocampus did not change significantly in aged animals. Our results indicate that abnormalities of NMDA receptor regulation by glycine recognition sites play an important role in degeneration of neuroni during aging process. Less