| 研究課題/領域番号 |
17K14946
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| 研究機関 | 九州大学 |
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研究代表者 |
LEIWE MARCUS 九州大学, 医学研究院, 助教 (80722008)
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| 研究期間 (年度) |
2017-04-01 – 2019-03-31
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| キーワード | 発生 / 発達 / Olfactory System / Optogenetics |
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| 研究実績の概要 |
Owing to the lab move which commenced in April 2017 we switched priorities and began by trying to alter mitral cell pruning using optogenetics.
Firstly, we devised a means of surgically implanting a small LED onto the LED. However, we discovered that these pups were rejected by their mother or the wires were bitten regardless of how careful the surgery was performed. This forced us to develop a means of hand rearing neonatal mice, determining the optimum food to feed, the frequency of feeding, temperature (of both the "nest" and LED) and humidity.
After finally determining the optimum parameters we could then assess the effect of maintaining the P1 synchrony levels from late postnatal day 2 (P2) to P6. Unfortunately, no differences were observed at either P6 or at P5. We are currently evaluating whether there may be a slight delay at P4. Additionally, we are evaluating whether there are sufficient levels of rhodopsin for chronic stimulation by doing long term stimulation ex-vivo and using electrophysiology.
Regarding the development of olfactory responses, our mice colonies have been established at the new lab. With a better 2 photon microscope in order to image at a faster frame rate. However, this switch in equipment required us to design and construct a new stage, integrate our olfactometer. Which we have achieved. Finally, we are currently constructing a respiration monitor that is capable of recording from neonatal mice.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
The unforeseen difficulties with the optogenetic experiments necessitated a much more intensive experimental protocol, with regular feeding every 2 hours for 3-4 days. Several trials were needed to optimise the aspects such as the concentration of the milk, and the nest temperature. We have now reached a point where the weight gain between hand- and maternally reared pups is equal, which indicates that development is unaffected by our methodology. However, to date we have been unable to determine any delays in the pruning process after optogenetic stimulation. Therefore, we have had to spend more time investigating different periods of chronic stimulation etc...
This meant we were unable to devote as much time to other aspects of the project as we would have liked. However, the other aspects are slowly coming online, so hopefully we will have completed them by the end of the next financial year. I am confident that we should be able to catch up to the deadline
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| 今後の研究の推進方策 |
As mentioned, we have switched priorities where the optogenetics were to be completed by the end of FY2017. However, this work has been more user intensive than anticipated and, as a result, has delayed our progress. However, if our current attempts at troubleshooting fail we have a back-up of using DAT-Cre;R26 TeTX mice to see if the removal of interneuron activity can change both the spontaneous activity and mitral cell pruning. This change in activity has already been established pharmacologically in the slice so it should finally indicate whether patterned activity is essential or not.
The measurement of phase coding in neonates has required us to develop a means of recording the respiration rate of neonatal mice, which is currently under construction. To speed up our efforts instead of a detailed study of the first post-natal work, the key stages of P2 (before pruning) and P6 (after pruning) will be investigated along with a juvenile (P21) and adult stage.
With regards to genetically modifying the phase code via interneurons, the both the DAT-Cre line and the R26-TeTX;Thy1-GCaMP6f line are being bred and so that aspect of the project is running on time.
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| 次年度使用額が生じた理由 |
Our priorities changed and we proceeded with altering mitral cell pruning first, which was cheaper than establishing the in-vivo set up.
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