Molecular basis of the circadian rhythm for peptides mRNA

在哺乳动物中，昼夜节律的起搏器被认为位于视交叉上核（SCN）。我们研究了大鼠SCN中所含肽的分子基础作用。采用ELA法测定光暗循环下SCN腹外侧区血管活性肠肽（VIP）和胃泌素释放肽（GRP）的共定位。在2天盲法大鼠中未观察到VIP和GRP的明显昼夜节律，但完整大鼠的VIP和GRP分别在主观夜间和白天显示出较高的浓度。用北方分析mRNA的表达，以了解这些肽合成的转录阶段。每天每4小时显微解剖4-6只脑的SCN，用酚-氯仿法提取总RNA。VIP mRNA水平在黑暗期间比在光照期间高。在皮层中没有观察到这种节律性。结果表明，神经肽可能参与昼夜节律属 ...更多信息 此外，为了了解这些肽类生物钟振荡的个体发生，我们对这些肽类生物钟振荡的发育过程进行了分析。VIP在黑暗期的浓度从纳塔尔后第4天开始高于光照期，并持续到断奶。为了建立一个长期观察SCN功能的体外系统，我们采用了一个分离的细胞培养系统来研究SCN的细胞机制。从光-暗周期下饲养的P-1日龄幼仔中获得SCN的分散细胞。测定了培养液中对SCN输出信号起重要作用的精氨酸加压素（AVP）的含量。这些细胞释放的AVP显示出准确的昼夜节律振荡。在光-暗周期下，当细胞制备的时相相差12小时时，AVP的昼夜节律发生了逆转。用ABC法对部分神经元进行AVP抗体标记。分散培养系统为阐明起搏机制提供了一个有用的模型。我们现在正在开发有机切片培养，它具有许多结构组织的优点。少

In mammals, a pacemaker of circadian rhythms is considered to be located in the suprachiasmatic nuclei(SCN). We examined the role of peptides contained in the SCN of rats for molecular bases. Vasoactive intestinal peptide (VIP) and gastrin releasing peptide are colocalized in ventrolateral area of the SCN were measured by ELA method under the light dark cycle. No clear circadian rhythm of VIP and GRP was not observed in the 2-days-blind rats, though the VIP and GRP in intact rats showed higher concentration during the subjective night and day, respectively. The Northern analysis of mRNA was done to know the transcriptional stage in the synthesis of these peptides The SCN of 4-6 brains were microdissected every 4hr throughout a day and total RNA was extracted by the phenol-chloroform method. VIP mRNA levels showed higher during the dark than in the light period. This rhythmicity was not observed in the cortex. The results suggest possible involvement of neuropeptides in circadian genera … More tion at their messenger RNA levels.Beside, to know the ontogeny of these peptides oscillation for circadian clock, the development of these peptides were analyzed. The concentration of VIP during the dark time began to higher in the dark period than in light one from the post natal day 4 and succeeded till the wean.To establish an in vitro system to observe the SCN function for a long duration, we used a dissociated cell culture system to study the cellular mechanisms of SCN. Dispersed cells of SCN were obtained from P-1 day pups whose mother were kept under light-dark cycle. Arginine-vasopressin (AVP), plays important role for the output signal form the SCN in vivo, in the culture medium were measured. The release of AVP from these cells showed a accurate circadian oscillation. The circadian rhythms of AVP were reversed in phase when the cell were prepared 12hr out of phase from the pups kept under the light-dark cycle. Some neurons were certified by AVP antibody using the ABC method. The dispersed culture system provides a useful model for elucidating the mechanism of pacemaker. We, now, are developing the Organotipic slice culture having a number of advantage of structural organization. Less