本研究致力於：（1）研發高通量微流控平臺實現斑馬魚幼蟲的長時間固定及培養；（2）研究觀 測未麻醉斑馬魚的心率、尾部和胸鰭擺動頻率在不同電刺激下的行為反應；以及（3）同時通過客 制雙光子顯微鏡獲取 GCaMP6 轉基因斑馬魚神經元放電模式鈣離子成像。相較於現有斑馬魚微流 控檢測裝置，我們可以準確系統地研究在平穩舒適環境下斑馬魚的電生理反應，而不會引入外部 幹擾刺激。 / 魚形設計實現了一批斑馬魚的精准固定。在該設計中，斑馬魚胸鰭及尾部活動被魚形微槽限制， 28.3 度的 E3 系統溶液持續不斷地流過斑馬魚鰓部確保所需的氧氣供應。為探究確定系統最優工作 條件以達到較小的流量壓差、更高的固定率及存活率，本工作進行了相應的理論推導及有限元建 模，去估測每個固定功能單元的壓力值、體積流速及流阻。在隨後 5 天觀測中，持續通入 2-7ml/h 的系統溶液，100%的斑馬魚可以存活並保持正常生存狀態，沒有焦躁不安和逃脫現象的出現。此 外通過逆轉流速的方向，被困斑馬魚能夠被很容易地釋放並收集，用於進一步的實驗。 / 為探究斑馬魚生理狀態：心跳、尾部及胸鰭擺動頻率，在不同流速或電刺激下，被長時間即時記 錄監測。為考慮由於發育階段不同而引入的生理狀態變化，我們同時也研究記錄了不同年齡斑馬 魚的心率改變。值得一提的是，我們運用脈衝電刺激進一步研究了斑馬魚在急性快速刺激下的恢 復能力和趨電性。實驗結果表明電激勵對行為響應有直接的刺激效應。並且長時間持續的高電壓 刺激可能會損傷斑馬魚運動協調能力甚至導致死亡。有趣的是， 我們觀測並研究了急促高脈衝電 壓誘發的具有高空間時間精度的癲癇模型。借助客制雙光子顯微鏡，我們進一步研究了在相應電 刺激下的神經信號處理過程的鈣離子成像。 / 該工作首次結合微流控晶片及雙光子顯微鏡成功研究了不同電刺激下斑馬魚受困於微流控裝置中 的生理狀態變化。我們的結果不僅論證了電刺激在調節斑馬魚生理行為反應的重要作用，同時也 為常見精神行為障礙疾病例如癲癇、阿爾茲海默症及帕金森症的未來治療研究提供了基於斑馬魚 疾病模型的實驗平臺 / This research aims to (1) develop a high-throughput microfluidic platform that enables long-term immobilization and culturing of zebrafish larvae; and (2) investigate behavioral responses of unanesthetized zebrafish, including heart beating rate, tail and fins flapping frequency, under different electric stimulations; and (3) simultaneously visualize neuron-firings pattern in GCaMP6 labeled zebrafish via a custom-built two-photon microscope. Compared with existing zebrafish microfluidic assays, this device enables precise and systematic study of electrophysiological reactions of zebrafish in a stress-free environment without external stimuli. / Precise motion constraint of zebrafish is realized through a fish-shaped design in which a batch of zebrafish is immobilized, with fins and tail constrained by the fish-shaped chambers and E3 medium continuously flowing through its gills at 28.3 ᴼC for oxygen supply. Analytic and finite element analysis models have been conducted to predict and evaluate the pressure, flow rate, and hydraulic impedance of each trapping unit to determine the optimal working conditions, achieving minimal pressure drop and high trapping efficiency. Experiments were performed over a period of 5 days under a flow rate of 2-7 ml/h. The results show a 100% survival rate; also, the zebrafish could maintain normal living condition with no signs of anxiety and escaping activities. By reversing the flow direction, the immobilized zebrafish can be easily released and recollected for further experiments. / To investigate the physiological states, i.e., heart pumping, tail and pectoral fins swing, of zebrafish larvae; experiments were performed under different flow rates and electric stimulations and recorded in real-time. To understand the physiological state variations at different developmental stages, the heart rates of zebrafish larvae at different ages were studied. In particular, pulse voltage treatment was applied to study the recovery capability of zebrafish from acute stimulation, and galvanotaxis. The results indicate that the electric treatment has a direct stimulatory effect on behavioral responses. It is also observed that continuous high-voltage stimulation may impair the motor coordination and eventually cause death of zebrafish. Interestingly, an acute high pulse voltage induced seizure model with spatiotemporal accuracy was observed and studied. By utilizing the custom-designed two-photon microscope and genetically labeled zebrafish, neuron signaling processes were visualized via Ca2+ imaging and corresponded to electrical stimulations. / This work, for the first time, successfully investigates physiological states variation of zebrafish larvae immobilized in microfluidic devices via electrical stimulations and multi-photon imaging. Our results not only demonstrate the critical role of electrical stimuli in regulating zebrafish larvae physiological behavioral responses, but also provides a platform for future therapeutic studies of common neurobehavioral disorders, e.g., epilepsy, Alzheimer and Parkinson diseases, using zebrafish model. / Chen, Wang. / Thesis M.Phil. Chinese University of Hong Kong 2016. / Includes bibliographical references (leaves ). / Abstracts also in Chinese. / Title from PDF title page (viewed on …). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_1292323 |
| Date | January 2016 |
| Contributors | Chen, Wang (author.), (thesis advisor.), Chinese University of Hong Kong Graduate School. Division of Mechanical and Automation Engineering. (degree granting institution.) |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography, text |
| Format | electronic resource, remote, 1 online resource ( leaves) : illustrations, computer, online resource |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons "Attribution-NonCommercial-NoDerivatives 4.0 International" License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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