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

本研究旨在研究大脑中兴奋性氨基酸受体的特征，主要集中在海马体和小脑。使用的方法是结合实验、体外受体放射自显影和受体蛋白的化学分析。NMDA受体正在形成一个由谷氨酸结合部位、甘氨酸结合部位、多胺结合部位和离子通道等多个亚组分组成的复杂结构的复合体。我们发现，Triton X-100预处理显著增强了NMDA识别位点的特异性结合。。该方法为阐明NMDA受体复合体的结合特性提供了可能。具体而言，甘氨酸、D-丝氨酸、6-7-二氯喹恶啉-2，3-二酮、7-氯尿酸(7-CKYNA)对N-甲基-D-天冬氨酸结合的抑制作用进行了实验研究，并对其抑制机理进行了分析。…更确凿的事实是，甘氨酸可以抑制NMDA结合。我们进行了7-氯尿酸(7-CIKYNA)对甘氨酸结合部位的识别实验，指出该物质可用于标记士的宁不敏感的甘氨酸结合部位。关于多胺结合部位，我们在海马区进行了H-亚精胺3的结合实验，发现多胺增强了H-MK 801的结合，并对二价阳离子如何结合进行了进一步的实验。对这些抑制实验的影响。在受体蛋白化学部分，对NMDA敏感的谷氨酸结合蛋白进行了溶解和部分纯化。纯化的蛋白质具有良好的结合能力。通过体外放射自显影定量研究大鼠脑内兴奋性氨基酸受体的增龄性变化。L-谷氨酸受体分为N-甲基-D-天冬氨酸型、奎斯奎宁型和海人酸型三种。其中，NMDA亚型在学习记忆和神经元呼叫死亡中起着特别重要的作用。在这项研究中，我们。重点研究了NMDA受体复合体。实验选用2月龄(青年)和21月龄(老年)Fischer 344系雄性大鼠。NMDA受体在海马CA1区密度最高，尤其是放射层和东方层。相邻锥体层的结合浓度相对较低。大脑皮层也含有高浓度的NMDA受体，并表现出区域性和层状变化。在尾壳核、嗅球、伏核、隔核、丘脑和杏仁核有中等水平的受体。中脑区和小脑皮质的受体水平较低。在NMDA受体复合体中，被H-甘氨酸标记的士的宁不敏感的甘氨酸受体在老年大鼠的斜脑区严重减少。在中脑区，与年龄平行的H-甘氨酸结合3S的减少仍然小于端脑。小脑中的II-甘氨酸结合部位没有明显变化。另一方面，H-CPP的结合部位在前脑区略有减少。老年动物海马区的H-CPP结合部位没有明显变化。我们的结果表明，甘氨酸识别部位对NMDA受体调节的异常在衰老过程中神经元的退行性变化起着重要作用。

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