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標題: | Carisbamate(RWJ-333369)在大鼠齒回抑制突觸前麩胺酸釋放 Carisbamate (RWJ-333369) Inhibited Presynaptic Glutamate Release in Rat Dentate Gyrus |
作者: | Chien-Cheng Shih 施建誠 |
指導教授: | 劉宏輝(Horng-Huei Liou) |
關鍵字: | 海馬,齒回,全細胞膜片箝制技術,突觸後興奮性電位, carisbamate,dentate gyrus,hippocampus,AMPA,NMDA,PPR, |
出版年 : | 2008 |
學位: | 碩士 |
摘要: | Carisbamate(RWJ-333369)為一新的神經作用藥物,在癲癇動物模型上展現廣效的抗癲癇作用,但其抗癲癇機轉仍未明。本論文研究carisbamate在大鼠海馬回齒回突觸的藥理作用,利用全細胞膜片箝制技術(whole-cell patch clamp)記錄齒回顆粒狀細胞(dentate granule cell)的突觸後興奮性電位(excitatory postsynaptic current)變化,評估carisbamate在本腦區對突觸傳導的影響。
本實驗使用年齡三週的大鼠所製備之冠狀腦切片,在齒回外側的molecular layer給予0.1 Hz的電刺激,於perforant pathway至齒回顆粒狀細胞的突觸前引發動作電位,並於突觸後顆粒狀細胞記錄glutamate活化α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)受體或N-methyl-D-aspartic acid(NMDA)受體所產生之電流。利用whole-cell patch clamp技術,記錄位於Wistar大鼠的hippocampus活體腦切片中DG的granule cell上由glutamate所引發的電流,藉由不同受體的阻斷劑,分離出AMPA受體和NMDA受體參與之電流並分析之。另外搭配不同的電生理技術,區分藥物的在突觸的作用位置。由於glutamate釋放受到鈣離子通道所調控,藉由同時投予鈣離子通道阻斷劑以及carisbamate藥物來評估鈣離子通道的作用。 結果顯示carisbamate對這兩類受體所引發之興奮性電位皆展現濃度依賴性的抑制效果;另外,100μM carisbamate會抑制AMPA受體在配對刺激(paired-pulse)下的性質,降低配對刺激比率(paired-pulse ratio)。30及100 μM 的carisbamate不會對胞外給予AMPA所引發之電流產生影響,也不會影響在TTX存在下的AMPA最小興奮性電流(miniature excitatory postsynaptic current)。另外,抑制L-type 鈣離子通道無法遮蔽carisbamate的抑制作用,顯示該藥物的抑制作用並非透過L-type 鈣離子通道。結果顯示(1)carisbamate確實會抑制glutamate受體所引發之興奮性電位並展現濃度依賴性的抑制作用。(2)carisbamate以一個相近的比例抑制AMPA或NMDA引發的興奮性電流,抑制配對刺激比率,不會影響胞外給予AMPA引發之電流,且對最小興奮性電流的電流大小不會產生抑制。因此,評估carisbamate的抑制作用來自抑制突觸前減少glutamate釋放。(3)carisbamate的抑制作用不是透過L-type 鈣離子通道而來。 DG長久以來和癲癇症,特別是顳葉癲癇症具有高度相關性。顳葉癲癇症會改變DG調控hippcampus的能力,使癲癇發作閾值降低。Glutamate為entorhinal cortex傳入DG的主要興奮性神經傳遞物質,carisbamate抑制突觸前glutamate釋放被視為抑制外來興奮性訊號的傳遞,減少外界興奮性刺激hippocampus,降低癲癇發作的機會。此突觸前的抑制作用被認為是carisbamate抗癲癇作用的之一。 Carisbamate (RWJ-333369) is a novel neuromodulator and shows a broad spectrum antiepileptic activity. We suggest that this neuroprotection effect might act through the regulation of excitatory synaptic transmission on DG. Our research evaluated its effect on excitatory signal transmission to dentate gyrus (DG) granule cell in rat hippocampus slice by whole-cell patch clamp technique. Evoked AMPA/NMDA receptor-mediated excitatory postsynaptic currents (eEPSCAMPA/NMDA) were recorded by whole-cell patch-clamp recording from the granule cells of DG in brain slice preparation of young Wistar rats (60-120 g, P21). We also used paired-pulse stimulation, exogenous AMPA application and AMPA receptor-mediated miniature EPSC (mEPSCAMPA) recording to evaluate the effects of carisbamate. Our results showed that carisbamate inhibited eEPSCAMPA/NMDA in a concentration-dependent manner. The decrease of paired-pulse ratio in 100 µM at 50 and 100 ms interval suggests that carisbamate acts presynaptically to reduce AMPA-receptor mediated current from perforant pathway. The response to exogenous AMPA application and mEPSC recording in the presence of TTX also showed that carisbamate had no effect on AMPA receptor. Furthermore, L-type Ca2+ channel blocker didn’t mask the reduction of eEPSCAMPA by 100 µM carisbamate. DG plays an important role in epilepsy, especially in temporal lobe epilepsy (TLE). The ability to regulate hippocampus is attenuated in TLE, which makes the seizure threshold lower. The inhibition of presynaptic glutamate release by carisbamate may contribute to its antiepileptic action. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/9775 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 藥理學科所 |
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