Matsushita T, Amagai Y, Terai K, Kojima T, Obinata M, Hashimoto S
Molecular Medicine Laboratories, Institute for Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585 Japan.
Neuroscience. 2006 Jul 7;140(3):849-56. doi: 10.1016/j.neuroscience.2006.02.059. Epub 2006 Apr 17.
The suprachiasmatic nucleus of the anterior hypothalamus is the center of an internal biological clock in mammals. Glutamate is the neurotransmitter of retino-hypothalamic tract responsible for mediating the circadian actions of light in rodents. N-methyl-d-aspartate receptors, particularly NR2B subunit are reported to be principally involved in photic resetting of the biological clock in vivo and in slice culture. But, the precise cellular mechanisms of the resetting are not elucidated, because no adequate neuronal cell lines derived from the suprachiasmatic nucleus have been established. We established a neuronal cell line, N14.5, derived from the suprachiasmatic nucleus of a transgenic rat harboring the temperature-sensitive simian virus 40 large T-antigen gene. When the cells were cultured at 39 degrees C, the morphological features were turned fibroblastic into neuronal round cell body with neurite extensions. These cells showed immunoreactivities for neuronal markers (betaIII-tubulin, microtubule-associated protein 2 and TAU2) and as well as for vasoactive intestinal peptide which is expressed in the ventrolateral region of the suprachiasmatic nucleus. The cells expressed N-methyl-d-aspartate receptors, particularly NR1 and NR2B subunits as revealed by quantitative PCR. N-methyl-d-aspartate activated phosphorylation of p44/42 mitogen-activated protein kinase and increased expression level of Per1 and Per2 mRNA. These results suggest that the N14.5 is a novel neuronal cell line derived from the ventrolateral region of the suprachiasmatic nucleus, and that N-methyl-d-aspartate receptors expressed in the cells are a functional receptor. The N14.5 cells may be a useful tool to elucidate numerous chronobiological processes, especially resetting mechanism induced by an external light signal.
下丘脑前部的视交叉上核是哺乳动物体内生物钟的中心。谷氨酸是视网膜下丘脑束的神经递质，负责介导啮齿动物中光的昼夜节律作用。据报道，N-甲基-D-天冬氨酸受体，特别是NR2B亚基，主要参与体内和切片培养中生物钟的光重置。但是，由于尚未建立源自视交叉上核的合适神经元细胞系，因此重置的精确细胞机制尚未阐明。我们建立了一种神经元细胞系N14.5，它源自携带温度敏感型猿猴病毒40大T抗原基因的转基因大鼠的视交叉上核。当细胞在39摄氏度下培养时，其形态特征从成纤维细胞样转变为具有神经突延伸的神经元圆形细胞体。这些细胞对神经元标记物（βIII-微管蛋白、微管相关蛋白2和TAU2）以及对视交叉上核腹外侧区域表达的血管活性肠肽具有免疫反应性。定量PCR显示，这些细胞表达N-甲基-D-天冬氨酸受体，特别是NR1和NR2B亚基。N-甲基-D-天冬氨酸激活了p44/42丝裂原活化蛋白激酶的磷酸化，并增加了Per1和Per2 mRNA的表达水平。这些结果表明，N14.5是一种源自视交叉上核腹外侧区域的新型神经元细胞系，并且细胞中表达的N-甲基-D-天冬氨酸受体是一种功能性受体。N14.5细胞可能是阐明众多生物钟生物学过程，特别是由外部光信号诱导的重置机制的有用工具。
