Molecular basis of the circadian rhythm for peptides mRNA

在哺乳动物中，昼夜节律起搏器被认为位于视交叉上核（SCN）。我们检查了大鼠SCN中所含肽的分子碱基的作用。在明暗循环下，用ELA法测定了血管活性肠肽（VIP）和胃泌素释放肽在SCN腹外侧区共定位的情况。2天盲大鼠未见VIP和GRP明显的昼夜节律，而完好大鼠的VIP和GRP分别在主观夜间和白天有较高的浓度。在一天中，每隔4小时对4-6只脑的SCN进行显微解剖，并用苯酚-氯仿法提取总RNA。VIP mRNA水平在黑暗期高于光照期。这种节律性在大脑皮层中没有观察到。结果表明，神经肽可能参与了昼夜节律的发生，更多的是在它们的信使RNA水平。此外，为了了解这些肽的生物钟振荡的成因，分析了这些肽的发育。暗期VIP浓度从出生后第4天开始在暗期高于光期，并一直持续到断奶。为了建立一个长期观察SCN功能的体外系统，我们使用分离细胞培养系统来研究SCN的细胞机制。将母鼠置于光照-黑暗循环中培养P-1日龄幼崽，获得分散的SCN细胞。对体内SCN输出信号起重要作用的精氨酸-抗利尿素（AVP）在培养液中的含量进行了测定。AVP的释放表现出精确的昼夜节律振荡。在光-暗循环条件下，在异期12小时制备AVP细胞时，AVP的昼夜节律发生了反转。用ABC法对部分神经元进行AVP抗体鉴定。分散培养系统为阐明起搏器的机制提供了一个有用的模型。目前，我们正在开发具有许多结构组织优势的有机切片培养。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