| Project/Area Number |
23K27147
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| Project/Area Number (Other) |
23H02454 (2023)
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Multi-year Fund (2024)
Single-year Grants (2023) |
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| Section | 一般 |
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| Review Section |
Basic Section 43040:Biophysics-related
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| Research Institution | Okinawa Institute of Science and Technology Graduate University |
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Principal Investigator |
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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| Project Period (FY) |
2023-04-01 – 2026-03-31
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| Project Status |
Granted (Fiscal Year 2024)
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| Budget Amount *help |
¥18,720,000 (Direct Cost: ¥14,400,000、Indirect Cost: ¥4,320,000)
Fiscal Year 2025: ¥3,120,000 (Direct Cost: ¥2,400,000、Indirect Cost: ¥720,000)
Fiscal Year 2024: ¥8,450,000 (Direct Cost: ¥6,500,000、Indirect Cost: ¥1,950,000)
Fiscal Year 2023: ¥7,150,000 (Direct Cost: ¥5,500,000、Indirect Cost: ¥1,650,000)
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| Keywords | voltage imaging / genetic targeting / 2-photon microscopy / in vivo / cerebellum / ANNINE-6 / Purkinje neuron / population activity / voltage-sensitive dyes / HaloTag |
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| Outline of Research at the Start |
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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| Outline of Annual Research Achievements |
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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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
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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| Strategy for Future Research Activity |
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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