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Eur J Neurosci. 2015 Nov;42(9):2678-89. doi: 10.1111/ejn.13061. Epub 2015 Oct 5.

Spatiotemporal profiles of arginine vasopressin transcription in cultured suprachiasmatic nucleus.

Author information

1
Photonic Bioimaging Section, Hokkaido University Graduate School of Medicine, Sapporo, 060-8638, Japan.
2
Department of Chronomedicine, Hokkaido University Graduate School of Medicine, Sapporo, 060-8638, Japan.
3
Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Kagawa, 761-0395, Japan.
4
Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Saitama, 332-0012, Japan.
5
Department of Regulatory Physiology, Dokkyo Medical University School of Medicine, Tochigi, 321-0293, Japan.
6
Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata, 951-8585, Japan.

Abstract

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.

KEYWORDS:

arginine vasopressin-Emerald-luciferase; bioluminescence; circadian rhythm; mouse; suprachiasmatic nucleus

PMID:
26342201
DOI:
10.1111/ejn.13061
[Indexed for MEDLINE]
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