2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38602An adequate amount of sleep is of paramount importance for a vigorous and productive lifestyle. In subjects suffering from sleep deprivation there is a desynchronization of the homeostatic neuronal regulation, which unavoidably leads to cognitive and metabolic disturbances. It has been reported that about 78% of the adult Taiwanese have suffered from chronic sleep disorders, and long-term sleeplessness will contribute to increased rates of traffic accidents and occupational injuries mainly resulted from memory deficiency and is thus of great social and economical importance. Previous electrophysiological and pharmacological studies have indicated that memory consolidation was performed in the hippocampus. The hippocampus is located on the medial surface of the temporal lobe and is received lots of cholinergic and serotonergic inputs from a variety of cortical regions as well as brainstem reticular nuclei. It has been reported that the interaction between cholinergic and serotonergic pathways might be particularly important for the regulation of long-term memory. Molecular evidences also suggested that hippocampal long-term potentiation might serve as the basic neuronal mechanism for developing the memory consolidation. The calcium-mediated nitric oxide synthase (NOS) activation through NMDA receptor opening would trigger numerous biochemical reactions involving in formation of hippocampal synaptic plasticity that is responsible for the consolidation of memory. Given that sleep deprivation would interrupt the homeostasis of neuronal communication and significantly disrupt the memorial cognitive function, the present study is aimed to determine the related neurochemical (along with their receptors) changes and the intracellular calcium distribution within the hippocampus of total sleep deprived rats. In the same time, the electron microscopy study together with the spatial memory and learning tests were also performed to extensively clarify whether the synaptic ultrastructural changes and behavioral alterations would significantly correlate to neurochemical expressions. Finally, with the viewpoint that melatonin is a powerful neuroprotective agent beneficial to reduce the oxidative stress in hippocampal neurons suffering from sleep deprivation, the present study is also design to explore the possible neuroprotective effects of this indole in improving the memory consolidation and neuronal plasticity. By deeply examining the neuronal mechanisms underlying the memory consolidation processes of sleep deprived rats will not only contribute to our better understanding of sleep loss related memory dysfunction, but also provide us a key insight into the pathogenesis of chronic cognitive disorders. In this regard, the first year of the present project will design to examine the detrimental effects of sleep deprivation on synaptic ultrastructural changes as well as cholinergic / serotonergic afferents related neurotransmitter expressions in the hippocampal regions. Based on the novel findings obtained from the first year, the second year of the present project will focus on detecting the effects of sleep deprivation on long-term potentiation relevant intracellular calcium distribution, glutamate expression, NMDA receptor activation, and neuronal NOS reactivity by the use of time-of-flight secondary ion mass spectrometry (TOF-SIMS) combined with quantitative immunohistochemistry. In the last year of the present project, different doses of melatonin will be given intraperitoneally during the sleep deprivation period to clearly clarify the advanced effect of melatonin on synaptic integrity, calcium current regulation and metabolic functions. By smoothly pursuing the present study, we can not only provide the detailed morphological, molecular and behavioral evidence which can help us to better understand the neuronal basis of memory consolidation, but also greatly increase our knowledge about the advanced effect of melatonin on improving the memorial function following sleep deprivation. With the gradually processing of this three-year project, we believe that our novel findings will contribute to the development of sleep medicine and offer the possibility for therapeutic use of melatonin in improving or restoring the functional integrity of memory following severe sleep disturbances.良好且充足的睡眠是維持活躍且具生產力生活品質之重要條件。處於睡眠剝奪環境 之個體將嚴重干擾神經系統之恆定運作，致使認知與代謝功能異常產生。據統計，台 灣成年族群中，約有78% 人口遭受慢性睡眠障礙之苦，而長期且慢性睡眠不足將干 擾記憶功能，導致交通事故及職業傷害等意外事故發生機率大增，嚴重影響整體社會 暨經濟成本。先前電生理與藥理學的研究已顯示，中樞神經系統之海馬回(hippocampus) 為職司記憶固化歷程(memory consolidation) 之重要單元。海馬回位於顳葉內側 (medial temporal lobe)，接收多數皮質區(cortical regions) 暨腦幹網狀區相關神經核 (brainstem reticular nuclei) 之膽鹼性(cholinergic) 暨血清素性(serotonergic) 投射。研 究指出，膽鹼性與血清素性相關神經傳導物質之交互作用可影響海馬回細胞之功能活 性(functional activity)，並在長期記憶(long-term memory) 形成過程中，扮演重要的角 色。此外，分子生物學的研究證據亦指出，海馬回中長期增益效應(long-term potentiation) 之訊息傳遞過程，適是記憶固化歷程之神經分子學基礎。由NMDA 受器 誘發之鈣離子(calcium) 媒介一氧化氮合成酶(nitric oxide synthase, NOS) 活化，可激 發諸多生化反應，胞內眾多生化反應進行可參與突觸塑性形成(formation of synaptic plasticity) 之調控，並進而導致記憶固化歷程發生。有鑑於睡眠剝奪將嚴重干擾神經傳 導物質之合成與聯繫，並對記憶功能產生破壞性影響，本研究之主要目的即旨在闡明 睡眠剝奪處理後，海馬回相關神經傳導物質(暨其接受器) 之活性表現與鈣離子濃度分 布之潛在改變。研究同時利用電子顯微鏡觀察(electron microscopy study) 並結合水迷 宮行為分析技術(water maze analysis)，深度探討海馬回細胞突觸之超微結構變化 (synaptic ultrastructural changes) 與空間記憶學習能力(spatial memory and learning ability) 之潛在關連。另一方面，為進一步探討褪黑激素(melatonin) 是否在記憶固化 歷程中，亦具明顯之神經保護效果(neuroprotective effects)，本實驗進而在睡眠剝奪實 驗鼠上施加不同劑量之褪黑激素，並檢視其海馬回內突觸超微結構、相關離子、神經 化學物質暨記憶學習能力方面之顯著差別。透過對睡眠剝奪環境下記憶固化歷程神經 機制之深入探討，將可有效增進吾人對睡眠促進記憶過程之了解，並對闡明慢性睡眠 障礙之記憶缺失衍生過程有所貢獻。緣此，本計畫第一年度擬利用電子顯微鏡法、定 量免疫組織化學法並結合水迷宮行為分析觀察，詳細探討實驗鼠經睡眠剝奪處理後， 海馬回細胞突觸超微結構變化、膽鹼性暨血清素性重要神經傳導物質活性表現及空間 記憶與學習能力之改變情形。計畫第二年度將以第一年度所得成果為基礎，擴大並深 入探討參與海馬回長期增益效應相關之鈣離子與麩胺酸、NMDA 受器暨一氧化氮合成 酶於睡眠剝奪之劇烈影響下，活性表現暨濃度分布之表現情形。本年度研究將使用飛 行式二次離子質譜儀(time-of-flight secondary ion mass spectrometry, TOF-SIMS) 進行 分子影像分析，並結合免疫組織化學反應，詳盡闡明睡眠剝奪對長期增益效應中， NMDA 受器與鈣離子流(calcium current) 暨一氧化氮合成酶之潛在影響。計畫第三年 度則將持續沿用前二年度之先進實驗方法，對睡眠剝奪鼠施予不同劑量之褪黑激素注 射，以釐清褪黑激素是否具改善神經塑性與增進記憶固化能力之神經保護功能。藉由 此三年期計畫的順利推動與執行，將提供完整且詳實的形態學、分子影像學暨行為科 學證據，除增進吾人對記憶固化歷程相關神經運作機制之認識外，亦提升學界對睡眠 剝奪造成記憶功能異常與認知障礙相關衍生過程之了解。此外，透過本研究計畫的逐 年進行，我們相信所得之研究成果亦將對臨床上使用褪黑激素治療或改善記憶缺失等 慢性睡眠障礙，有顯著之參考價值與貢獻。Project