共同研究報告書


研究区分 一般研究

研究課題

 哺乳類冬眠中の脳内熱産生メカニズムの光学的解析
新規・継続の別 継続（R05年度から）
研究代表者／所属 自然科学研究機構生命探究センター
研究代表者／職名 JSPS Postdoc
研究代表者／氏名 張 菁圃

研究分担者／氏名／所属／職名
　
氏　　名
所　　属
職　　名

1

 李明亮 自然科学研究機構生命探究センター 特任助教

2

 榎木亮介 自然科学研究機構生命探究センター 准教授

3

 山口良文 北大低温研

研究目的 Hibernators use the strategy to survive harsh environments， such as low temperature and food scarcity， by reducing metabolism and body temperature. Body temperature is mainly controlled by brown adipose tissue (BAT) that is rich in mitochondria and highly expresses uncoupling protein 1 (UCP1)， where heat is generated. I found that UCP1 is also expressed in the hamster brain. However， it is unclear whether the brain UCP1 is functional. We will measure the intracellular temperature and study the role of brain UCP1 in the thermoregulation and the physiological functions during hibernation.
　　
研究内容・成果 We’ve confirmed the expression of UCP1 in neurons in the paraventricular nucleus of the hypothalamus (PVN) and in astrocytes near the third ventricle， which is innervated by tanycytes， in hamster， but not in mouse， with immunohistochemical staining. We next performed quantitative analysis to characterize the expression of brain UCP1 through the hibernation. We found that hypothalamic UCP1 expression increased during the chronic cold exposure and returned to the same level as that of warm control during the deep torpor， whereas the amount of BAT UCP1 peaked during the deep torpor. It suggested a BAT independent thermoregulatory mechanism in the brain.
To study the thermogenic capabilities of hypothalamic astrocyte and neuron， we conducted the acute calcium imaging from hamster brain slice and found that both astrocyte and neuron elicited stimuli-dependent activity to the cold stimulation. Then， we will measure the intracellular temperature by delivering the genetically encoded temperature indicator (GETI) B-gTEMP into the cytosol and mitochondria of hypothalamic astrocyte or neuron in Syrian hamsters with adeno-associated virus transfection. The temperature inside cytoplasm and mitochondria will be monitored in acute brain slice under multi-photon microscopy with different temperatures ranging (5- 35 degrees). We have installed and optimized the temperature control system. Next，we’ve established the virus injection techniques and confirmed the expression of B-gTEMP in the hamster brain. To further understand the physiological role of UCP1 in real hibernation，we will perform in vivo fibre photometry recording from hypothalamic astrocyte and neuron expressing B-gTEMP during hibernation. To this aim，we have constructed the fibre photometry systems and established operations for long-term recording.
To check the function of hypothalamic UCP1，we will use shRNAi to knockout UCP1 and examinate the uncoupling property from acute brain slice. We’ve designed the shRNA and prepared the platform. To specify the necessity of UCP1，all the experiments will be conducted in both wild-type and UCP1 knockout (KO) hamsters. The UCP1-KO hamsters were successfully generated by Prof. Yamaguchi at the Institute of Low Temperature Science (ILTS) at Hokkaido University， and the brain samples were collected and the expression of UCP1 was tested by immunostaining.
　　
成果となる論文・学会発表等 [1] CHANG Ching-Pu， YAMAGUCHI Yoshifumi， WATANABE Masatomo， NEMOTO Tomomi， ENOKI Ryosuke. “Identification of uncoupling protein 1 (UCP1) in the hypothalamus of Syrian hamster brain”. Abstract for “The 46th Annual Meeting of the Japan Neuroscience Society. Sendai， Japan. August 1-4， 2023.”

[2] CHANG Ching-Pu. “Optical Analysis of Brain Thermogenesis in Mammalian Hibernator.” Oral present for “Hibernation Biology Meeting in Sapporo and Jozankei Onsen. October 11-12， 2023”