| 研究課題/領域番号 |
26285161
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| 研究機関 | 筑波大学 |
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研究代表者 |
Pavlides C 筑波大学, 人間系, 教授 (50712808)
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| 研究分担者 |
小川 園子 筑波大学, 人間系, 教授 (50396610)
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| 研究期間 (年度) |
2014-04-01 – 2017-03-31
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| キーワード | sleep / memory / hippocampus / amygdala / fear conditioning |
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| 研究実績の概要 |
In an initial study, we demonstrated that pharmacological inhibition/activation of PKA in the hippocampus during sleep produced a deficit/enhancement in long-term fear memory (Cho, et al., in preparation). In 2015 we undertook a set of experiments to determine the time course of PKA activation in sleep. Rats were fear conditioned and either allowed to sleep between 0-4 or 4-8h post conditioning or were kept awake. Upon reaching sleep criteria of 30min (or at a similar time for the awake group), the animals were quickly sacrificed, their brains dissected and the ratio of PKA/pPKA were calculated using Western blot analysis. We found that fear conditioning produced an enhancement in the PKA/pPKA ratio, however, regardless of sleep time after conditioning. (The findings were presented at the Physiological Society of Japan; Pavlides, et al. 2016). In a separate set of experiments we are testing the hypothesis that sharp wave ripples, seen in the hippocampal EEG during sleep, may be a physiological mechanism via which PKA is activated for the consolidation of memory. Following fear conditioning, animals are allowed to sleep. Sharp waves ripples are detected and in one group of animals stimulation to hippocampal afferents is applied to suppress their activity, or in controls stimulation is applied outside of ripple activity. These experiments are ongoing and the findings have been submitted as an abstract for the Japan Neuroscience Society (Sypniewski, et al. 2016).
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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
We have made a great deal of progress in the proposed studies. First, we have set up a separate fear conditioning apparatus that will enable us to perform the experiments more efficiently. The experiments to determine PKA levels in sleep did not produce conclusive results - a trend towards a higher PKA/pPKA ratio in fear conditioned animals was observed, however, regardless of sleep or time after conditioning. Two possible reasons for this could be sleep quality (previously we used only observation) and brain section extraction (brains were sectioned into various relatively large regions under visual observation). We are currently repeating the experiments with the addition of EEG/EMG recordings to ensure sleep levels, and are also using a 'brain punch' method for brain sample collection to ensure we are getting precise brain regions (e.g., dorsal/ventral hippocampal CA1/CA3, DG, amygdala, etc.). For the studies aimed at determining the role of sharp wave ripples on PKA activation, we have installed a dedicated surgery/electrode implantation setup as well as a sleep recording chamber. In preliminary studies we developed methods for recording sharp wave ripples as well as tested various stimulation parameters for the elimination of ripple activity. These experiments are now underway for fear conditioning, sleep recording/stimulation and sample collection for Western blot analysis for determining effects of sharp waves on PKA activation.
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| 今後の研究の推進方策 |
Thus far, we have used a contextual fear conditioning paradigm in which both the hippocampus and amygdala are involved. In future experiments, we will test differential amygdala PKA activation using a cued fear conditioning paradigm, for which the amygdala is involved but not the hippocampus. These results will further solidify the role of PKA activation in sleep for the consolidation of long-term memories. Besides PKA, a number of other genes (e.g., MAPK; PI3K) are also involved in fear memory consolidation. In the current funding period studies we will use pharmacological methods to suppress these gene cascades specifically in sleep to determine effects on long-term memory.
Given positive results in the experiments investigating the role of sharp wave ripples in PKA expression, we will then proceed to determine the effects of PKA and sharp wave ripples in sleep in behavioral experiments. More specifically, we will investigate whether suppression of sharp wave ripples disrupts long-term fear memory and whether pharmacological manipulation of PKA can reverse the memory deficits. These experiments will directly test the hypothesis that sharp wave ripples produce their effects via PKA activation.
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| 次年度使用額が生じた理由 |
A main reason that not all the funds have been used, thus far, is that we planned to build electrophysiological equipment along with purchasing software for the recording of sleep behaviors. These are custom build, which are not guaranteed to function without testing. Thus, initially a setup was built and is being tested before more elaborate and duplicate equipment is ordered. The drugs being used are expensive, but do not have an infinite shelf life. Once we have ensured that the drugs are working, more of these will be ordered. Initially, a person who could perform surgeries, was not identified. Currently, such a person has joined the team and is being paid a part-time salary. During this year we plan to publish some of the results, which will also require publication fees, etc.
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| 次年度使用額の使用計画 |
As discussed above, during the 2016 budget year, we will be purchasing further recording equipment, including commutators and cables, testing cages, fear conditioning setup, etc, to speed up the collection of data. This will also require the purchase of more animals, histology supplies, etc. Given the additional results from the ongoing studies, publication fees, presentations at meetings, etc. will also be increased.
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