林豊益Lin, Li-Yih黃順志Huang, Shun-Chih2019-09-05不公開2019-09-052018http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22G060543020S%22.&%22.id.&http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/103940氨(包含氣態的NH3以及離子態的NH4+)是魚類代謝的主要廢物。魚體內氨濃度提高會造成魚隻游泳不平衡、內分泌失調、離子調節功能失常甚至死亡。魚類的側線系統對於感測水流的方向非常重要，影響著魚的其他行為，如：游泳的平衡、逆流行為(rheotaxis)、逃跑以及捕食獵物。側線神經丘(neuromast)是由許多毛細胞(hair cell)所組成，可感測機械性的水流刺激。當水流撥打毛細胞上的纖毛束，刺激纖毛束頂端的機械性通道mechanotransducer (MET) channel開啟，使鈣離子流入毛細胞而產生水流方向的訊息。本研究利用斑馬魚仔魚作為實驗物種，研究離子態的氨是否會影響到斑馬魚側線毛細胞的功能，進而使其逆流行為表現不正常。在本研究中，將斑馬魚仔魚浸泡於氯化銨的水溶液30分鐘後，分析其逆流行為、游泳速度、毛細胞數目(免疫染色及FM1-43染色)以及毛細胞的鈣離子流量。實驗結果發現，在斑馬魚仔魚的逆流行為以及游泳速率，皆會顯著下降。然而免疫染色及FM1-43染色結果卻顯示毛細胞數目並不會減少。離子流量部分，毛細胞的鈣離子流量受到抑制，同時也在測量到銨離子流入毛細胞，但是使用MET通道的抑制劑新黴素及鑭離子，可以降低銨離子的流入。綜合以上實驗結果，我們的研究第一次發現：氨由MET通道進入毛細胞，影響側線毛細胞的功能，進而可能對斑馬魚仔魚的逆流與游泳行為造成影響。 關鍵字：氨、側線、逆流行為(rheotaxis)、神經丘(neuromast)、毛細胞、MET通道Ammonia (including NH3 and NH4+) is the major nitrogenous waste of fish. Accumulation of ammonia in fish can lead to swimming imbalance, disorder of endocrine, disruption of ionic balance and even death. The lateral line system of fish plays a critical role in sensing the direction of water flow, which is required for several behaviors including swimming balance, rheotaxis, escape and predation. The lateral line neuromast is composed of several mechanosensory hair cells which can sense water flow through the deflection of cilia on the top of hair cells. As water deflects the cilia, a mechanotransducer channel (MET channel) on cilia opens and allows Ca2+ to flow into the hair cells and finally generates a sensory signal. By using zebrafish larvae as a model, this study attempted to investigate whether ionic NH4+ can impair the function of lateral line hair cells and cause abnormal rheotaxis behavior. After exposing to NH4Cl for 30 min, the rheotaxis behavior, swimming velocity, number of hair cell (labeled by FM1-43 and immunostaining), and MET channel-mediated Ca2+ influx was analyzed. The NH4+ treatment significantly suppressed the rheotaxis behavior and swimming velocity. Although the number of hair cells did not change, the Ca2+ influx was significantly suppressed after NH4+ treatment. In addition, the NH4+ influx at hair cells was also found in fish exposed to NH4+ and the influx was blocked by the MET channel blockers, suggesting that NH4+ could be absorbed by hair cells through the MET channel. Taken together, this study shows for the first time that NH4+ exposure can impair the function of the lateral line hair cells in zebrafish larvae and the MET channel in hair cells is the site for the NH4+ entrance. Keywords：ammonia, lateral line, rheotaxis, neuromast, hair cell, MET channel氨側線逆流行為神經丘毛細胞MET通道ammonialateral linerheotaxisneuromasthair cellMET channel