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Neuro Imaging

開催日 2014/9/11
時間 10:00 - 11:00
会場 Room H(304)
Chairperson(s) 喜多村 和郎 / Kazuo Kitamura (東京大学大学院医学系研究科 神経生理学 / Department of Neurophysiology, Graduate School of Medicine, The University of Tokyo, Japan)
今井 猛 / Takeshi Imai (理化学研究所 発生・再生科学総合研究センター(理研CDB) / RIKEN Center for Developmental Biology, Japan)

Imaging CREB phosphorylation in live mouse brain using split luciferase

  • O1-H-2-4
  • 石本 哲也 / Tetsuya Ishimoto:1 眞野 寛生 / Hiroki Mano:1 森 寿 / Hisashi Mori:1 
  • 1:富山大院・医薬・分子神経 / Dept Mol Neurosci, Grad Sch Med Pharm Sci, Univ of Toyama, Toyama, Japan 

Phosphorylation of cAMP response element binding protein (CREB) and subsequent gene expression are thought to be key events in memory formation and recovery process from depression. Therefore the development of the method for analyzing the CREB phosphorylation in live cells or animals is demanded. We employed the split luciferase technique to monitor the phosphorylation of CREB in live cells. In this technique, firefly luciferase was cleaved into N-terminal and C-terminal segments. Kinase inducible domain of CREB and its interacting domain of CREB binding protein (CBP) were fused with N-terminal and C-terminal segments of luciferase respectively. By the interaction between these two fusion proteins, split segments complement each other to be a functional luciferase that can emit light. The photon from probe proteins was increased in response to forskolin treatment that up-regulated cAMP in the cells. This increase was cancelled by replacement of serine 133 to alanine in CREB phosphorylation domain in the probe protein. Next, we generated the novel transgenic mouse strain that expressed the probes and succeeded in monitoring light emission from brain of awake mice. We observed increased light emission from cerebral cortex after the acute treatment of imipramine, a tricyclic antidepressant. Increase in CREB phosphorylation by same treatment in cerebral cortex was confirmed using western blotting. These results demonstrate this transgenic mouse strain can be used for the imaging of CREB phosphorylation in live mouse brain. Furthermore, we also show the changes in pattern of CREB phosphorylation after reserpine treatment that is known to induce depression-like symptoms in mouse.

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