研究生: |
陳郁嵐 Chen, Yu-Lan |
---|---|
論文名稱: |
利用離體電生理模式探討斑馬魚端腦雙側之功能性連結及突觸可塑性 Study of the functional connectivity and synaptic plasticity in bilateral telencephalon of zebrafish by using in vitro electrophysiological approaches |
指導教授: |
呂國棟
Lu, Kwok-Tung |
學位類別: |
碩士 Master |
系所名稱: |
生命科學系 Department of Life Science |
論文出版年: | 2016 |
畢業學年度: | 104 |
語文別: | 英文 |
論文頁數: | 78 |
中文關鍵詞: | 斑馬魚 、腦側化 、端腦 、突觸可塑性 、長期增益效應 、長期抑制效應 、NMDA受器 、同側 、對側 、電生理 |
英文關鍵詞: | cerebral lateralization, contralateral, electrophysiological techniques, ipsilateral, NMDA receptor |
DOI URL: | https://doi.org/10.6345/NTNU202205103 |
論文種類: | 學術論文 |
相關次數: | 點閱:184 下載:10 |
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腦側化(cerebral lateralization)為一種脊椎動物常見的現象,意指大腦兩側半球各自對不同的功能扮演較優勢的角色(dominant);腦側化對於生物個體的行為表徵扮演著重要的角色,例如人類的左右腦各負責不同類型的工作;又或是魚類會利用兩眼視覺系統,區別熟悉與陌生的環境。硬骨魚的端腦(telencephalon)被認為與學習和記憶的形成有關,特別是端腦的背外側區(dorsal lateral, Dl)及背中側區(dorsal medial, Dm)最為相關,前人利用螢光染劑DiI注射於Dl腦區後,可在Dm腦區偵測到螢光訊號,證明了Dl和Dm腦區間存在神經投射的連結。近年來,斑馬魚已成為探討學習與記憶、藥物成癮以及焦慮等行為非常重要的模式物種。從斑馬魚的胚胎發育研究以及行為觀察,已有充分的證據顯示斑馬魚腦部和哺乳類一樣,具有腦側化的現象,但關於斑馬魚端腦腦側化的研究卻非常缺乏。因此,本實驗目的為利用電生理技術探討傳遞到同側(ipsilateral)以及對側(contralateral)的Dl-Dm投射路徑的神經傳遞與突觸可塑性(synaptic plasticity)現象的異同。首先,實驗測得在單側的Dl給予電刺激,能夠在同側以及對側的Dm引發一個負電位的電場電位(negative field potential),還可利用高頻電刺激(high frequency stimulation, HFS) 及低頻電刺激(low frequency stimulation, LFS),分別誘發出長期增益效應(long-term potentiation, LTP)以及長期抑制效應(long-term depression, LTD) ,這兩項均為探討神經突觸可塑性的重要性指標。實驗中利用連續五次的HFS (每秒100 Hz)來誘發LTP,結果顯示在同側及對側的Dm腦區所誘發的LTP現象,此外,如預先投予NMDA受器的拮抗劑D-AP5 (30 μM)10分鐘後才進行誘發,可完全阻斷對側LTP的產生。但將D-AP5藉由灌流廓清後,則又可重新利用HFS誘發出LTP。由此證實了HFS所誘發的對側LTP,需依賴NMDA受器的活化。將左右兩側誘發的LTP實驗結果,經交叉比對分析後發現,從左側及右側的Dl給予HFS,所誘發出的同側LTP會有所差異,從右側誘發出的LTP強度會比左側誘發的小,而單從右側Dl誘發的LTP而言,其訊號強度同側會較對側的小。本實驗接着以LFS (每秒1 Hz)持續刺激20分鐘,或是投予代謝型谷氨酸受體興奮劑DHPG (40 μM) 10分鐘來誘發LTD,兩者都能誘發出至少維持一小時的LTD現象。而我們將以代謝型谷氨酸受體興奮劑DHPG所誘發的同側及對側結果比較後,我們發現對側的抑制效果較同側好。另外,Dm腦區所誘發的電場電位可以分成突觸的(synaptic, P1)以及非突觸的(non-synaptic, N2)組成,而對側Dm腦區的P1時間較同側Dm腦區的時間長,可能造成的原因為對側端腦的紀錄點較同側端腦紀錄點的距離更遠,因此有對側P1時間較同側長的現象。另外,藉由即時聚合酶鏈鎖反應(Real-time polymerase chain reaction, Real-time PCR)技術,我們發現NMDAR1a受器的mRNA在左側端腦的表現上高於右側端腦的趨勢。綜合而言,本研究的重要發現為首次觀察到斑馬魚端腦的Dl腦區和Dm腦區之間,存在著同側以及對側神經連結的突觸可塑性現象,並運用了電生理模式證明了斑馬魚端腦具有腦側化的現象。本研究成果將有助於日後為利用基因轉殖斑馬魚探討端腦腦側化分子機轉奠定基礎。
Cerebral lateralization is a common feature among vertebrates including reptiles, fishes and amphibians. It is a phenomenon of specialization of function between right and left hemisphere of the brain. The complementary of function between right and left hemisphere leaves individual a more integrated cognitive behavior, which is profoundly affected by lateralization. For example, the preferential eye use of zebrafish. Zebrafish tends to use particular eye to observe different stimulus and is known as left eye system (LES) and right eye system (RES) which resemble two hemisphere of human brain each responsible for different tasks. In teleost, telencephalon is considered related to the limbic system of mammals, which plays essential role on the learning and memory, especially lateral (Dl) and medial (Dm) division of the dorsal telencephalon. Tract-tracing studies suggested the neural connection between Dl and Dm division via afferent Dl fibers projected to Dm division.
Zebrafish has becoming an important animal model for studying the neural mechanism of learning and memory, drug addiction, and anxiety disorder. Our previous results showed the phenomenon of cerebral lateralization in zebrafish. The present study was aimed to investigate neurotransmission and synaptic plasticity in projections from the Dl to the Dm in zebrafish by using electrophysiological approaches. Our results showed that giving unilateral electrical stimulation at either side of the Dl division would evoke a negative field potential (FP) in both contralateral and ipsilateral side of Dm division. We also conducted the test of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD). By giving five trains of high frequency stimulation (HFS; 100 Hz for 1 s), we induced NMDAR-dependent LTP. To further investigate whether HFS-induced LTP is NMDAR-dependent, we apply NMDA receptor antagonist, DL-AP5 (30 μM, suprafused for 10 mins) that completely blocked the HFS-induced LTP in both side of Dm. In addition, the formation of LTP restored after washout DL-AP5 by continuous ACSF suprafusion. It proved the involvement of NMDA receptor in the LTP formation. We also demonstrated a significant difference on the stimulation site and the amplitude of LTP. Electrical stimulating from right side of Dl would bring out smaller amplitude of LTP compared with stimulating from the left. Furthermore, stimulation from the right will bring out smaller amplitude of LTP on the ipsilateral side than contralateral side. Collectively, these results suggest a cerebral lateralization existed in the Dm-Dl circuit of zebrafish. We also inducted LTD by giving low frequency stimulation (LFS; 1 Hz for 1 s) or applying group I mGluR agonist, DHPG (40 μM, suprafused for 10 min). Both of them successfully induced contralateral LTD that can last for at least 1 hr. We discovered the PS amplitude of DHPG-induced LTD on contralateral side was smaller than that of ipsilateral side. Another finding in our experiment was Dm field potential can be divide into synaptic (N2) and non-synaptic (P1) components. The latency of initial positive deflection of contralateral Dm lasted longer than ipsilateral Dm might be caused by the different distance of stimuli through biological tissue towards recording cathode between contralateral and ipsilateral side. Furthermore, by real-time polymerase chain reaction (real-time PCR), we observed the tendency of higher NMDAR1a mRNA expression in the left telencephalon than in right telencephalon. In conclusion, our results suggested that the connection between Dl-Dm divisions in the telencephalon of zebrafish possess synaptic plasticity, and the feasibility of using electrophysiological techniques to study neural mechanisms underlying cerebral lateralization in zebrafish.
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