概日リズムを外部コントロールし生活リズムを調節する研究
Rika Numano1, 2*, Minako Matsuo1, Naofumi Kimura1
1,21,
沼野 利佳 * ,松尾 美奈子 木村 尚文
1 Environmental and Life Science and EIIRIS) Toyohashi University of Technology

豊橋技術科学大学応用化学生命生命系1・エレクトロニクス先端融合研究所2･Light stimulus

Introduction

Analysis

Measurement

SCN

SCN

Living things on the earth have internal clocks with about
24-hour period, which autonomously drive their
physiology and behavior and reset their phase ofcircadian rhythms by environmental cues. Central
circadian clock in mammals is located in thesuprachiasmatic nuclei (SCN) in the hypothalamus.
The SCN functions symmetry
to the left-right axis of the bodySCN
to keep normal circadian rhythms.
However, what a mechanism toPacemaker
Outputsynchronize symmetry in the SCN
has not been elucidated in detail.

Measurement of the GFP signal

Photo-stimulation only on left side

ResultRight (Non photo-stimulated)

Left (photo-stimulated)

Input

Material and Method

Fluorescence signal (Per1 expression)can be induced Fluorescence can be induced
fast in the part closer to the photo-stimulated side and the photo-stimulated side immediatelyslow in the part far from the photo-stimulated side(within 3hr) after photo-stimulation.

trans-MAG

Fluorescence signal can be induced also in the non photo-stimulated part (NPS)
after adding AVP inhibitor in contrast to not after adding VIP inhibitor .

cis-MAG405 nmPer1 expression level･Light stimulation system
The photoreactive compound MAG to operate ion channelof glutamate receptor by UV and visible light.
Azobenzene

GFP signal in right (Non photo-stimulated) after some kinds of inhibitor

Per1 expression level

In this study, we observed the Per1 expressional induction
by photo-stimulation in Per1 :: GFP transgenic (Tg) mouse.
Only the left side of the SCN slice in Tg was photoactivated by a photo-reactive compounds NEO-MAG. GFP
signal was compared between left and right part of the SCN
slice, which was cut at the third ventricle of the median line
to amputate contact of nerve fibers

Ca+Na+

Elapsed time after light stimulation

Elapsed time after light stimulation

Maleimide

Glutamic acid

488 nmAntagonist

The NEO-MAG can phto-stimulate the local area of brainslice by laser irradiation in wildtype.
･Sample preparation
GFP
Per1::GFP Per1promoter

Excised brain

Per1::GFPTransgenic mice

After adding AVP inhibitor

After adding VIP inhibitor

Create a brain slice

Cut the SCN in themidline of chiasma

Dropping a light reactive compound・Clock gene Period1(Per1) shows daily expressional rhythms in theSCN to regulate circadian rhythms. Per1 expression is induced in theSCN about 30 to 90 minutes after the light stimulus to the eye to reset
the phase of circadian rhythms.

chemicalf.c.

Hederagenin

3μM○AVP（AVPV1a) receptor antagonist

SR49059

1 μM

×

GRP receptor antagonist

RD176252

0.4μM

×

VIP（VPAC2)receptor antagonist

Effect in the NPS

The signals to reset the circadian rhythms is transmitted in the SCN for right-left functional
synchronization by not AVP（AVPV1a) and GRP, but by VIP（VPAC2) and GABAa receptor .

Conclusion

1) Even after cutting the SCN slices in the midline of chiasma toblock the contact of nerve fibers, the Per1 expressional induction
was also observed in the non-photostimulated side of the SCN
as well as photo-stimulated side.
Per1 induction could be monitored by GFP signal in Per1:GFP Tg.
2) It is possible that not only by nerve contact but also by the1 Left part of the SCN was photo-stimulated by a 405 nm and 488 nm
humoral factors, the signals to reset the circadian rhythms islaser, after adding NEO-MAG chemicals and cutting off the slice in ZT16.
transmitted in the SCN for right-left functional synchronization
2 Real-time observation of GFP signal in the SCN slice of Per1 :GFP Tgby VIP and GABAa receptor.
mice was performed in once every 20 minutes for 8 hours.