| 研究課題/領域番号 |
23K27147
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| 補助金の研究課題番号 |
23H02454 (2023)
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| 研究種目 |
基盤研究(B)
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| 配分区分 | 基金 (2024)
補助金 (2023) |
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| 応募区分 | 一般 |
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| 審査区分 |
小区分43040:生物物理学関連
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| 研究機関 | 沖縄科学技術大学院大学 |
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研究代表者 |
Kuhn Bernd 沖縄科学技術大学院大学, 光学ニューロイメージングユニット, 教授 (90599557)
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| 研究期間 (年度) |
2023-04-01 – 2026-03-31
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| 研究課題ステータス |
交付 (2024年度)
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| 配分額 *注記 |
18,720千円 (直接経費: 14,400千円、間接経費: 4,320千円)
2025年度: 3,120千円 (直接経費: 2,400千円、間接経費: 720千円)
2024年度: 8,450千円 (直接経費: 6,500千円、間接経費: 1,950千円)
2023年度: 7,150千円 (直接経費: 5,500千円、間接経費: 1,650千円)
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| キーワード | voltage imaging / genetic targeting / 2-photon microscopy / in vivo / cerebellum / ANNINE-6 / Purkinje neuron / population activity / voltage-sensitive dyes / HaloTag |
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| 研究開始時の研究の概要 |
Overall, this project is multidisciplinary and several people with different background work together. We hope that we can image voltage from single neurons in the behaving mouse in the near future to study neuronal networks on a millisecond time scale.
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| 研究実績の概要 |
In FY2023, we started our project which consists of 4 parts: (1) Synthesizing voltage-sensitive ANNINE dyes which can be used to label genetically targeted neurons in vivo. (2) Genetically tagging specific neurons in vivo. (3) Show cell-type specific labeling of neurons with the voltage-sensitive ANNINE dye. (4) In vivo imaging of neuronal voltage changes with cellular resolution at kHz temporal resolution. Regarding (1), we synthesized the first ANNINE-6 dyes with phosphate groups to label neurons by expressing a phosphatase. However, the novel dyes turned out to be too hydrophobic. We are still synthesizing new, more hydrophilic ANNINE dyes with phosphate groups. Additionally, we also try to add a HaloTag ligand to ANNINE-6. The first ANNINE dyes with HaloTag ligand look very promising but still need further optimization. Regarding (2), we have now Adeno Associated Viruses to express phosphatase or HaloTag on the cell surface. Both systems were tested in vivo. Regarding (3), We performed test experiments with a HaloTag ligand attached to fluorescein which works beautifully. We still synthesize better ANNINE dyes. Regarding (4), we prepared the behavioral stage for in vivo experiment and installed a high speed camera which allows us to image neuronal activity at up to 5 kHz. We currently image Ca2+ activity at high temporal resolution in vivo.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
Part (2) and (4) of the project progressed very smoothly. Also, the synthesis of ANNINE-6 with phosphate groups and with HaloTag ligand (1) were successful but need fine tuning. This fine tuning and the synthesis of different variants is a necessary process and was expected to take time. Testing of the new dyes in vivo is done as soon as new dyes are synthesized.
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| 今後の研究の推進方策 |
We will progress as planned. Most importantly we will work on part (1), the synthesis of ANNINE dyes with phosphate groups and HaloTag ligand. Part (2) is completed. We work on part (3) whenever new ANNINE dye synthesis are completed. While new dyes are synthesized, we image neuronal activity with Ca2+ indicators at high speed and establish data analysis pipelines. Thereby, we will test the system and the system will be ready to go when one of the cell-type specific ANNINE-labeling methods works.
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