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研究生:李淨芬
研究生(外文):Ching-Fen Lee
論文名稱:探討恐懼記憶形成之分子機轉
論文名稱(外文):Studies on the molecular mechanisms of fear-potentiated startle
指導教授:簡伯武簡伯武引用關係
指導教授(外文):Po-Wu Gean
學位類別:碩士
校院名稱:國立成功大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:105
中文關鍵詞:恐懼記憶
外文關鍵詞:synapsin Ifear-potentiated startle
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許多文獻指出,大腦中的杏仁核是誘導恐懼記憶形成與表現的重要調控樞紐。關於恐懼記憶形成的機轉之探討,本實驗室對於突觸後的訊息傳遞路徑已有深入的瞭解,然而對於突觸前機轉的瞭解確是有限,因此在本研究裡遂想回過頭來加以探討在恐懼狀況下,恐懼記憶形成的突觸前機轉。已知在恐懼狀況 ( fear-conditioning ) 下,位於大腦中的杏仁核會誘導出相關連性的長期增益現象 ( associative long-term portentiation ) ,並且也知道蛋白質的磷酸化對於長期的突觸可塑性 ( long-term synaptic plasticity ) 而言是一個強而有力的調節者。在本研究裡所要探討的蛋白質磷酸化現象是synapsin I。已經知道synapsin I大量地存在於神經末稍內並且平時黏附於包囊( vesicles ) 上的外圍。synapsin I的磷酸化能夠調控包囊從「不容易釋放後備區」( reserve pool ) 釋放出來而漸漸地靠向active zone,進而釋放出神經傳遞物質,如glutamate。在本研究中也就是要探討恐懼記憶的形成與synapsin I磷酸化的相關性,並以synapsin I的磷酸化作為神經傳遞物質釋放的一個指標。由實驗結果顯示,在恐懼狀況下,synapsin I的磷酸化確實有顯著的增加並且可以維持至30分鐘。先前本實驗室已證實出:投與group II metabotropic glutamate receptor agonist ( L-CCG ) 能夠減少突觸前神經傳遞物質的釋放而導致恐懼記憶的形成;因此進一步地使用DCG-IV試劑,發現能夠抑制在恐懼狀況下synapsin I的磷酸化;緊接著觀察到DCG-IV的作用能藉由其拮抗劑 ( LY341495 ) 而逆轉。更進一步地,觀察恐懼記憶的消除 ( extinction ) 對synapsin I磷酸化的影響;由實驗結果發現,將老鼠的恐懼記憶消除後,synapsin I的磷酸化受到抑制。在本研究裡最後也探討在恐懼狀況下,synapsin I的一些磷酸化位置的表現情形,並進一步觀察這些磷酸化的抑制是否影響恐懼記憶的形成。由實驗結果發現,在恐懼狀況下,synapsin I的PKA phosphorylation site、CaMKII phosphorylation site 以及cdk5 phosphorylation site等位置的表現情形可藉由其kinase inhibitor而抑制,而當這些phosphorylation被抑制了之後,恐懼記憶也隨之被抑制。
Accumulated evidence indicate that the amygdala is a crucial neuronal locus for the induction and expression of fear memory. Fear conditioning induces associative long-term potentiation in the amygdala and protein phosphorylation is a powerful regulator of long-term synaptic plasticity. Protein phosphorylation is also a direct and rapid modulator of exocytosis in neurons. Previous studies indicate that protein kinases play an important role in mediating the release of synaptic vesicles from the reserve pool and the synaptic-vesicle-associated phosphoprotein, synapsin I, is a key kinase substrate in this process. Hence, my study is to prove whether fear conditioning induces phosphorylation of synapsin I and whether this effect is subjected to regulate by memory extinction. In this study, I have shown that synapsin I is phosphorylated after fear training and phosphorylated synapsin I is subsequently dephosphorylated when animals receive extinction training.
Previous studies in our laboratory demonstrated that L-CCG-induced long-term depotentiation ( LTD ) leads to a long-lasting decrease in transmitter release which was induced by presynaptically group II metabotropic glutamate receptor ( mGluRII )-mediated inhibition of Ca2+-sensitive adenylyl cyclase, resulting in a decrease in cAMP formation and PKA activation. Therefore, I used the mGluRII agonist, (2S, 2R,3R)-2-(2,3-Dicarboxycyclopropyl)glycine ( DCG-IV ), to examine its effect on the phosphorylation of synapsin I and used the antagonist, LY341495, to test whether it could reverse the effect of DCG-IV. The results revealed that DCG-IV decreased the phosphorylation of synapsin I that was reversed by LY341495 .
Finally, I determined the different phosphorylation site of synapsin I in fear conditioning and found that the phosphorylation of PKA/CaMKI phosphorylation site, CaMKII phosphorylation site, and cdk5 phosphorylation site were increased following fear conditioning. By using the specific kinase inhibitor to block synapsin I phosphorylation, I found that these inhibitors reduced the phosphorylation of different synapsin I phosphorylation site and inhibited the fear-potentiated startle.
中文摘要 1
英文摘要 4
縮寫檢索表 6
第一章 緒論 8
第二章 材料與方法 25
第三章 結果一:探討Synapsin I 的磷酸化對於恐懼記憶形成的影響 42
第四章 結果二:探討在恐懼狀況下Synapsin I的不同磷酸化位置之表現並進而利用激酶抑制劑抑制恐懼記憶的形成 56
第五章 討論 71
第六章 建立另一個恐懼老鼠的動物模式 76
第七章 結論 85
參考文獻 89
圖表索引 102
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