跨突觸分子探針研發 – 偵測果蠅大腦中神經活性

Development of Trans-synaptic Probes for Detecting Synapses Functional Connectivity in Drosophila Brain

陳思妤(Sih-Yu Chen)

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(2015年)

摘要

化學突觸為神經元藉由傳導物質遞訊息的位置。了能以視覺方法觀測經傳導物質釋放的突觸位置，我們在先前研究中已修改了統綠色螢光蛋白跨突觸重組技術，製造出新的蛋白質分子探針並且已用於偵測果蠅功能性。然而，此綠色螢光重組探針仍然不足以讓我們分辨神經傳導是否具有活性為了能更趨近動態與活性的目，我們嘗試發展具有光轉換特螢蛋白探針。理想上已形成的 突觸能以重組螢光方式被觀測，並且在特定波長激發下改變原有螢光顏色。這樣的改變可視為重設基準，因若當組已被原來的螢光顏色又重新被觀察到就意味著此突觸持續不斷形成、尚未轉換分子探針。近來，我們嘗試以 PSmOrange2這個光轉換螢蛋白作為新型跨突觸分 子探針的核心。在幾次試誤後，我們企圖以一個新切割位置分開 PSmOrange2並且引入到跨突觸分子探針設計中。藉由把前、後的同時表現在小鼠神經瘤細胞 (N2a) 細胞中，我們已確認了探針的螢光重組和轉換能 力。我們正在 製造能表現此跨突觸光轉換螢蛋白探針的基因殖果蠅，並以做為研究神經

關鍵字

神經突觸 ； 分子探針 ； 綠螢光蛋白重組 ； 神經傳導

並列摘要

Chemical synapses are specialized gaps where neurons transmit signals through neurotransmitter to each other or to non-neuronal cells. To visualize the incidence of functional synaptic transmission, we have modified the GFP reconstitution across synaptic partners (GRASP) and created chimeric trans-synaptic protein probes that are able to label the functional connectivity in Drosophila in previous work. However, our trans-synaptic probes are not sufficient to discriminate whether the signals are observed from the active-neurotransmissions or already established neuron connections. In order to purely detect the synaptic transmission in a dynamic and activity-dependent fashion, we attempt to create a more powerful tool by constructing split-photoconvertible fluorescent protein in the probe design instead of stable GFP. By changing the color of already established neural connections, we are able to see neurons that are still actively transmitting signals. In our work, we adopted the PSmOrange2 to construct the new trans-synaptic probes system. To optimize the probes, we have tested different modifications and confirmed the properties of reconstitution and photoconverting in N2a cell culture system. We are now operating microinjection to create transgenic flies expressing the trans-synaptic probes so that the neurotransmission can be observed in vivo.

並列關鍵字

synapses ； molecular probes ； GRASP ； neural transmission

參考文獻

Evan H. Feinberg, M. K. V., 2 Andres Bendesky, 1 George Wang, 2 Richard D. Fetter, 3 Kang Shen, 2, *, & 1, a. C. I. B. (2007). GFP Reconstitution Across Synaptic Partners (GRASP) Defines Cell Contacts and Synapses in Living Nervous Systems. Neuron.

Fan, J.-Y., a, b., , Z.-Q. C., a, , H.-P. W., a, . . . *. (2007). Split mCherry as a new red bimolecular fluorescence complementation system for visualizing protein–protein interactions in living cells. Biochemical and Biophysical Research Communications.

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