“突觸”一詞最早出自1897年由生理學家Michael Foster編撰的Textbook of Physiology（《生理學教科書》），源於希臘語Synapsis，意思是“連線”。現代神經科學將突觸定義為神經元之間或神經元與效應細胞之間相互接觸並實現功能聯絡的節點。當神經衝動到達上一級神經元（突觸前神經元）的軸突終末時，觸發神經遞質的釋放，這些神經遞質穿過突觸間隙作用到突觸後膜受體，將神經訊號傳遞給下一級神經元（突觸後神經元）。因此，突觸後膜受體在突觸傳遞過程中發揮訊號“接收器”的關鍵作用。透過構建特異的膜受體標記策略，結合熒光視覺化技術，定位和實時追蹤特定膜受體動態變化規律，是理解突觸生理功能以及突觸異常相關神經和精神疾病機制的首要任務。

Neuroscience Bulletin在2021年第2期以封面文章形式發表了上海交通大學醫學院徐天樂教授團隊的技術方法論文Postsynaptic Targeting and Mobility of Membrane Surface-Localized hASIC1a。封面圖中，孫悟空的火眼金睛代表高效特異的神經元膜表面hASIC1a標記與視覺化工具，識破白骨精的偽裝代表對hASIC1a時空特異性的突觸定位和動態膜轉運規律的揭示。

突觸，這一高度特化且複雜的亞細胞結構承載著神經細胞間的資訊傳遞，是腦功能和整體行為的基礎[1-3]。突觸前、後膜遍佈數量種類龐雜的受體和離子通道，這些膜蛋白的精確定位和動態調節是神經元執行資訊輸入、加工和傳導的關鍵。而受體和離子通道的定位或數量調控異常是多種神經和精神性疾病的潛在機制[3, 4]。由此，神經元如何維持突觸受體和離子通道的動態平衡是神經科學領域的重要科學問題。

圖1 穀氨酸受體的膜轉運與突觸可塑性

（修改自Gambino F, Choquet D. Eyes Wide Open on AMPAR Trafficking during Motor Learning. Neuron. 2020）

膜受體或離子通道主要在細胞表面發揮功能。對於穀氨酸受體的突觸定位和膜轉運已經有比較系統的認識[5-7]。大量研究揭示穀氨酸受體突觸定位和膜轉運決定興奮性突觸傳遞、突觸長時程增強（Long-term potentiation, LTP）與長時程抑制（Long-term depression, LTD）以及學習記憶等腦認知功能（圖1）[8-11]。有趣的是，近年發現酸敏感離子通道1a（Acid-sensing ion channel 1a, ASIC1a）也參與突觸傳遞和可塑性[12-17]。ASIC家族主要由4個基因（ACCN1，ACCN2，ACCN3，ACCN4）編碼6種亞基（ASIC1a，ASIC1b，ASIC2a，ASIC2b，ASIC3，ASIC4），在腦中ASIC1a是通道功能的核心亞基。三聚體離子通道（ASIC4除外）感受胞外H+升高而開放，主要引起Na+和Ca2+內流，貢獻學習記憶、慢性痛和負性情緒等生理和病理過程（圖2）。但ASIC1a調節興奮性突觸功能的具體機制尚不清楚。通常認為突觸小泡與突觸前膜融合，H⁺和穀氨酸被一同釋放到突觸間隙，啟用位於突觸後膜的ASIC1a，從而貢獻突觸傳遞和可塑性，並調節學習記憶等相關行為（圖2）[14, 18-21]。

圖2 ASIC1a的結構功能

（修改自Jasti J et al. Structure of acidsensing ion channel 1 at 1.9 A resolution and low pH. Nature.2007和Kreple CJ et al. Drug abuse andthe simplest neurotransmitter. ACS Chem Neurosci. 2014）

然而，作為一種非經典突觸後膜受體，神經元膜表面功能性ASIC1a的突觸定位和膜轉運規律仍不清楚，主要原因在於缺乏用於細胞和組織免疫染色的ASIC1a特異性抗體等標記工具。雖然小鼠（mASIC1a）和人源ASIC1a（hASIC1a）序列同源性高達98%，但兩者在膜表面丰度和通道特性等方面存在差異，而對hASIC1a的研究更顯不足[22-24]。

徐天樂教授團隊與合作者綜合分子克隆、單細胞電生理、噬菌體展示、免疫染色和活細胞成像、單分子示蹤等手段，篩選hASIC1a胞外域位點以插入標記序列（hASIC1a-298HA299或hASIC1a-298pHluorin299）和從噬菌體組合抗體庫中篩選特異識別hASIC1a胞外域構象的抗體[25]（ASC06-IgG1或ASC06-IgG1-Alexa488），開發了細胞膜表面功能性hASIC1a的標記和視覺化工具，用於固定狀態下研究神經元表面hASIC1a的亞細胞定位和活細胞實時觀察樹突上hASIC1a的動態轉運（圖3）。

hASIC1a分佈於皮層神經元胞體和樹突，尤其在突觸後膜富集；活細胞及單分子水平觀察到樹突上hASIC1a的正向插膜和側向遷移，外源給予腦源性營養因子（Brain-derived neurotrophic factor, BDNF）可以促進hASIC1a的膜表達和側向運動以及靶向突觸的膜轉運（圖4）。這項研究為ASICs領域提供了關鍵的標記工具和技術支援，揭示了hASIC1a在神經元細胞膜表面的分佈和運動規律，提示hASIC1a時空轉運在突觸功能調節中的關鍵作用。

圖3 神經元細胞膜表面hASIC1a的標記與視覺化。

hASIC1a的胞外域抗體ASC06-IgG1或HA標籤用於固定狀態下神經元膜表面hASIC1a的標記和定位分析（I）；胞外域pHluorin標籤用於活細胞狀態下實時觀測突觸表面的hASIC1a動態變化（II）；熒光基團修飾抗體ASC06-IgG1-Alexa 488用於單分子或單顆粒水平研究樹突表面hASIC1a的側向運動（III）。

未來利用該工具，藉助人幹細胞分化神經元和hASIC1a基因敲入小鼠，結合超高分辨和在體雙光子成像，在更精細的水平研究內源hASIC1a的突觸定位和膜轉運規律，探究hASIC1a的動態調控和突觸結構功能以及動物行為的關聯，最終解析H+/ASICs參與學習記憶等腦功能的新機制，甚至在整體水平靶向ASIC1a膜轉運實現對慢性痛、恐懼和焦慮的干預。

圖4 BDNF調控hASIC1a的動態膜轉運

熒光漂白恢復實驗表明BDNF促進ASIC1a靶向樹突棘的正向膜轉運；單分子示蹤實驗提示BDNF加速樹突表面hASIC1a沿著細胞膜的側向遷移。

詳情：

Song XL, Liu DS, Qiang M, Li Q, Liu MG, Li WG, et al. Postsynaptic targetingand mobility of membrane surface-localized hASIC1a. Neurosci Bull 2021, 37:145-165.

https://link.springer.com/article/10.1007/s12264-020-00581-9

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