臺灣博碩士論文加值系統

在哺乳動物，下視丘的視叉上核(suprachiasmatic nucleus, SCN)是控制日週期(circadian rhythm)的主要樞紐。在in vitro腦切片的狀況下，視叉上核神經元的自發性動作電位也有24小時的週期性變化。麩胺酸(glutamate)是視叉上核主要的興奮性神經傳導物質，可以調控光線對生物時鐘的重設(entrainment)，而且會相移視叉上核的神經活性週期。為了了解麩胺酸對於視叉上核神經元電生理的影響，我分別利用了三種實驗方法來探討這個主題，分成：1. 細胞外記錄實驗，在腦切片內記錄視叉上核神經元的自發性動作電位頻率。2. blind patch全細胞電流箝制術實驗，記錄細胞膜電位的變化。3. 「小塊組織」(mini-explant)全細胞電壓箝制術實驗，記錄麩胺酸對於視叉上核神經元離子電流的作用，這個「小塊組織」實驗法也是本實驗室自行發展出的新技術。實驗結果發現，麩胺酸會使視叉上核神經元的自發性動作電位頻率增加以及細胞膜電位去極化。接著在「小塊組織」的實驗中，則是第一次發現到，麩胺酸會抑制視叉上核的鉀離子電流。改變電壓會影響麩胺酸抑制鉀離子電流的作用，表示這個抑制作用是具有電壓依賴性的。若將細胞外溶液換成無鈣溶液，並不會影響這個抑制作用，所以細胞外鈣離子的存在對於麩胺酸抑制鉀離子電流並不是必須的。而且，麩胺酸所抑制的電流有很大部分是對TEA敏感的鉀離子電流。由實驗結果以及前人研究可以推測得知，麩胺酸所抑制的電流應該是延遲整流性鉀離子電流。而且，麩胺酸的作用機轉以間接透過麩胺酸受器再去活化其他機制的可能性較高。根據這樣的實驗結果，可以推想的是，或許麩胺酸抑制延遲整流性鉀離子電流而改變動作電位的波形或頻率，進而影響視叉上核神經元的興奮性。

The suprachasmatic nucleus (SCN) of the hypothalamus is a major pacemaker for circadian rhythms in mammals. The spontaneous firing rate of SCN shows a spontaneous 24-h oscillation in vitro. Glutamate is the primary excitatory neurotransmitter that mediates photic entrainment and causes phase-shift of the neuronal activity rhythms. In this study, I use three different series of experiments to examine the effects of glutamate on the electrophysiological activities of the SCN neurons. The spontaneous firing rate and membrane potential of the SCN neurons in brain slices were investigated by extracellular recording and whole-cell current-clamp technique, respectively. Furthermore, the effects of glutamate on ionic currents in SCN neurons were examined with the whole-cell voltage-clamp technique in “mini-explant” method, a new method developed in our lab. Glutamate depolarizes SCN neurons and increases their firing rate. Moreover, glutamate inhibits potassium current in SCN neurons in “mini-explant” method. The inhibition was voltage-dependent and not influenced by extracellular calcium. The currents inhibited by glutamate are mostly TEA-sensitive. The results suggest that delayed rectifier potassium current is probably the major target of glutamate inhibition. The mechanisms of the glutamate inhibition may mediate through glutamate receptors, then activated others intracellular pathways. Inhibition of the delayed rectifier potassium current by glutamate could in turn influence the excitability of SCN neurons.

目錄
圖次………………………………………………….iv
中文摘要……………………………………………..v
英文摘要………………………………………….....vi
緒論…………………………………………………..1
材料與方法…………………………………………12
結果…………………………………………………17
討論…………………………………………………24
結論…………………………………………………31
參考文獻……………………………………………44
圖 次
圖一、視叉上核小塊組織示意圖…………………………………….32
圖二、小塊組織實驗中所記錄的電流-電壓關係圖…………………33
圖三、細胞外記錄：麩胺酸使視叉上核神經元自發性動作電位頻率增加……………………………………………………………….34
圖四、比較不同濃度的麩胺酸造成自發性動作電位頻率增加的百分比……………………………………………………….………35
圖五、麩胺酸對於細胞膜電位的作用……………………………….36
圖六、麩胺酸抑制視叉上核神經元之鉀離子電流………………….37
圖七、麩胺酸的抑制作用並非電流本身的減少…………………….38
圖八、麩胺酸對鉀離子電流的抑制作用是不可逆的……………….39
圖九、比較不同測試電壓下，麩胺酸對鉀離子電流的作用……….40
圖十、在無鈣溶液下，麩胺酸對鉀離子電流的作用……………….41
圖十一、比較控制溶液以及無鈣溶液下，麩胺酸造成鉀離子電流的抑制比例………………………………………………………..42
圖十二、1mM TEA對麩胺酸抑制作用的影響……………………...43

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