Development of Trans-synaptic Probes- A Potential Approach for Detecting Both Synaptic Proximity and Function

神經突觸探針- 同時偵測神經突觸間距及功能的新方法

王育倫(Yu-Lun Wang)

指導教授 ： 桑自剛

國立清華大學/生命科學院/生物科技研究所/碩士(2011年)

https://doi.org/10.6843/NTHU.2011.00268

摘要

在神經系統中，突觸是位於軸突神經細胞末梢，用來將神經傳導物質從一個神經細胞傳遞到下一個神經細胞的地方。透過神經傳導物質由前突觸神經細胞釋放到突觸間隙，然後被後突觸神經細胞上的接受器所接收的過程，可以引發動作電位讓神經訊號傳遞到下一個神經元。這個釋放的過程主要透過突觸小泡的循環及一群SNARE蛋白來調控，其中Synaptotagmin I 是一種主要分布在突觸小泡上的v-SNARE蛋白，在它的羧基端由兩個可以感測鈣離子的C2 區域所構成。當接受到鈣離子的刺激，Synaptotagmin I C2 區域的兩個環狀結構 (L1及L3)會穿透細胞膜的磷脂雙層而幫助突觸小泡的融合。根據這點，我們利用蛋白質融合技術結合綠色螢光蛋白片段重組的概念製作出一對位於神經突觸兩端的探針，期望能在活體動物的腦中直接偵測神經傳導物質釋放的過程。在現階段的細胞實驗當中，我們將兩支探針分別表現在神經細胞及非神經細胞當中，然後在一起培養的狀態下發現綠色螢光蛋白片段可以進行重組；同時也在果蠅的神經系統中測試這組探針的可行性，這將會是一個可以同時偵測神經突觸的間距及功能的新方法。

關鍵字

神經突觸 ； 突觸間距 ； 突觸功能 ； 探針 ； 綠色螢光蛋白 ； 重組

並列摘要

In the nervous system, synapses are cellular junctions at axonal terminal that permit neurotransmitters to pass from a neuron to another. Neurotransmitters from presynaptic neurons are released to the synaptic cleft, and then accepted by receptors embedded on the postsynaptic neuron surface. Through this process, an evoked action potential relays the signal to the next cell. To empty the neurotransmitters in the synaptic vesicles, a group of SNARE proteins are required. Synaptotagmin I is a vesicular SNARE protein, which is mainly located on synaptic vesicles. It contains two independent C2-type Ca2+ sensing domains (called C2A and C2B) in the c-terminal. It has been reported that, in response to Ca2+, Synaptotagmin I utilizes two flexible loops (L1 and L3) of the C2 domain to partially penetrate the hydrophobic core of the lipid bilayer, thus intiate vesicle fusion. According to this finding, we are developing synaptotagmin I-based synaptic probe in conjuction with reconstitutive GFP, and aim to decode the process of neurotransimission in a visible means in living brain. In our pilot study, we expressed pre- and post-synaptic probes seperatly in two types of cells and found that the splitted GFP can be reconstituted in the co-culture system. We are currently testing these probes in the context of neurotransmission and in the Drosophila nervous system. The success of this approach may provide a novel way to detect both transynaptic proximity and function in vivo.

並列關鍵字

trans-synaptic ； synaptotagmin ； neuroligin ； neurexin ； GRASP ； co-culture

參考文獻

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