Yoshikawa Tomoko, Nakajima Yoshihiro, Yamada Yoshiko, Enoki Ryosuke, Watanabe Kazuto, Yamazaki Maya, Sakimura Kenji, Honma Sato, Honma Ken-ichi
Photonic Bioimaging Section, Hokkaido University Graduate School of Medicine, Sapporo, 060-8638, Japan.
Department of Chronomedicine, Hokkaido University Graduate School of Medicine, Sapporo, 060-8638, Japan.
Eur J Neurosci. 2015 Nov;42(9):2678-89. doi: 10.1111/ejn.13061. Epub 2015 Oct 5.
Arginine vasopressin (AVP), a major neuropeptide in the suprachiasmatic nucleus (SCN), is postulated to mediate the output of the circadian oscillation. Mice carrying a reporter gene of AVP transcription (AVP(ELuc)) were produced by knocking-in a cDNA of Emerald-luciferase (ELuc) in the translational initiation site. Homozygous mice did not survive beyond postnatal day 7. Using the heterozygous (AVP(ELuc/+)) mice, a bioluminescence reporter system was developed that enabled to monitor AVP transcription through AVP-ELuc measurement in real time for more than 10 cycles in the cultured brain slice. AVP(ELuc/+) mice showed circadian behaviour rhythms and light responsiveness indistinguishable from those of the wild-type. Robust circadian rhythms in AVP-ELuc were detected in the cultured SCN slice at a single cell as well as tissue levels. The circadian rhythm of the whole SCN slice was stable, with the peak at the mid-light phase of a light-dark cycle, while that of a single cell was more variable. By comparison, rhythmicity in the paraventricular nucleus and supraoptic nucleus in the hypothalamus was unstable and damped rapidly. Spatiotemporal profiles of AVP expression at the pixel level revealed significant circadian rhythms in the entire area of AVP-positive cells in the SCN, and at least two clusters that showed different circadian oscillations. Contour analysis of bioluminescence intensity in a cell-like region demonstrated the radiation area was almost identical to the cell size. This newly developed reporter system for AVP gene expression is a useful tool for the study of circadian rhythms.
精氨酸加压素（AVP）是视交叉上核（SCN）中的一种主要神经肽，据推测它介导昼夜节律振荡的输出。通过在翻译起始位点敲入翡翠荧光素酶（ELuc）的cDNA，制备了携带AVP转录报告基因（AVP(ELuc)）的小鼠。纯合子小鼠在出生后第7天之后无法存活。利用杂合子（AVP(ELuc/+)）小鼠，开发了一种生物发光报告系统，该系统能够通过在培养的脑片中测量AVP-ELuc实时监测AVP转录超过10个周期。AVP(ELuc/+)小鼠表现出与野生型无法区分的昼夜行为节律和光反应性。在培养的SCN切片的单细胞以及组织水平均检测到AVP-ELuc中强烈的昼夜节律。整个SCN切片的昼夜节律是稳定的，在明暗周期的光照中期达到峰值，而单细胞的昼夜节律则更具变异性。相比之下，下丘脑室旁核和视上核的节律性不稳定且迅速衰减。像素水平上AVP表达的时空分布显示，SCN中AVP阳性细胞的整个区域存在显著的昼夜节律，并且至少有两个簇表现出不同的昼夜振荡。对细胞样区域生物发光强度的轮廓分析表明，辐射区域几乎与细胞大小相同。这种新开发的AVP基因表达报告系统是研究昼夜节律的有用工具。
