利用腦側化反轉斑馬魚探討端腦的功能

Study of telencephalon function using cerebral lateralization inversed zebrafish

李宗祐(Li Zong-You)

指導教授 ： 呂國棟

國立臺灣師範大學/理學院/生命科學系/碩士(2021年)

https://doi.org/10.6345/NTNU202100408

摘要

腦側化(cerebral lateralization)是指左右邊大腦半球(cerebral hemisphere)各會偏重執行(dominate)某些特定功能，為一種脊椎動物常見的現象。這種功能性的腦側化(functional lateralization)又可追溯至左右腦結構，或是特定分子於左右腦分佈的不對稱性(asymmetric)。許多文獻指出上丘腦(epithalamus)是探討斑馬魚腦部結構的不對稱(structural asymmetry)之重要標的。副松果體位於(parapineal)上丘腦中，正常情況下，該核團約有98%的機率會位於左腦中，僅少部分「腦反轉」個體(brain inversed)其副松果體會位於右腦。本計劃比較一般個體及腦反轉個體間，外顯行為及兩側端腦功能差異，結果將有助於了解斑馬魚腦結構不對稱性對，於腦功能側化的影響。
目前已知功能性腦側化會體現於個體的認知(cognition)、情緒(emotion) 和學習與記憶(learning and memory)等面向。本研究利用腦側化反轉斑馬魚，探討整合結構不對稱性對情緒與認知的影響。本研究分別使用野生型(wild-type, WT)及foxd3:GFP品系的基因轉殖(transgenic, TG) 斑馬魚作為實驗對象，該TG品系斑馬魚的副松果體會表現外源性的綠色螢光蛋白(exogenic green fluorescence protein, GFP)，故可藉此判別副松果體位置，以篩選出正常(無反轉)的左側副松果體個體(left-side parapineal, Lpp)及腦反轉的右側副松果體個體(right-side parapineal, Rpp)個體。研究包括了四階段的行為實驗(behavioral experiment)，第一階段是基礎運動能力測試，用以確認各實驗動物的自發性游動(locomotor activity test)是否正常，基礎運動功能正常的個體，方能進入下一階段實驗。第二階段實驗為類焦慮行為(anxiety-like behavior)測試，本研究選用新穎性水箱測試(novel tank test)，交叉比對不同組別實驗動物的類焦慮行為表現。第三階段則為抑制性逃避學習測試(inhibitory avoidance test)，檢核腦反轉對恐懼記憶(fear memory)的建立是否有差異。第四階段則是利用腦反轉的TG斑馬魚，探討隨著腦構造反轉後，其功能性腦側化及偏重執行的外顯行為是否同樣出現反轉的現象。
實驗結果顯示，腦反轉個體(Rpp)的運動功能與學習能力未受影響，但類焦慮行為明顯增加。在認知功能方面，對無反轉斑馬魚(Lpp)施以右側端腦破壞，會干擾空間及恐懼學習能力，故推測其與野生型斑馬魚相同，空間及恐懼的學習主要由右側端腦所主導，而腦反轉的斑馬魚(Rpp)則轉變為左側端腦主導。因而可推論斑馬魚的功能性腦側化將隨腦部發育反轉，而發生左右顛倒的情形。我們相信端腦功能性側化確實存在於斑馬魚的學習和記憶過程中，但該現象是來自於先天形生(innated)的神經迴路？或是後天經驗學習後(acquired)才逐步建立？仍有賴進一步探討。本研究也觀察到即使對端腦進行較小面績的破壞，但倘若破壞到較關鍵的部位，仍會對空間和恐懼學習造成顯著的損害。

關鍵字

斑馬魚 ； 腦側化 ； 類焦慮行為 ； 學習與記憶 ； 副松果體

並列摘要

Cerebral lateralization is widely found in vertebrates. It is defined as a hemispheric preference in performing certain functions. Numerous studies reported that the parapineal gland, a nucleus of the epithalamus, is an essential target for exploring the structural and functional asymmetry of zebrafish's brain. Normally, approx. 98% of the zebrafishes, its parapineal glands appear in the left hemisphere. Previous studies also showed the cerebral lateralization is involved in sensory perception, emotion, learning, and memory. Our previous studies showed the spatial learning and memory was impaired after unilateral hemisphere lesion. These results evidence the functional dominance existed in the cerebral hemisphere of the zebrafishes.
A transgenic foxd3: GFP line was applied in the current study. It expresses an exogenic green fluorescence protein in the parapineal. Therefore, the location of the parapineal could be visualized and confirmed via fluorescence microscopy. The transgenic zebrafishes were then cataloged by its parapineal location to the left side parapineal group (Lpp) and the right side parapineal group (Rpp). A modified aspiration ablation paradigm was applied. Animals were subjected to the unilateral ablation then subjected to behavioral experiments five days after the ablation. There are four stages in the present study. The first stage was aimed to evaluate the motor function and spontaneous activities using locomotor activity monitoring. Abnormal zebrafishes were then excluded from the subsequent experiments. In the second stage, novel tank dive test was used to examine anxiety-like behavior exhibited in the wild type and foxd3: GFP zebrafishes. The cerebral lateralization effect on fear learning was studied via an inhibitory avoidance test in the third stage. The fourth stage was aimed to explore whether the functional brain lateralization of zebrafish with inversion of the brain structure also reverses in explicit behavior.
Results showed that neither motor function nor learning ability were impaired in the brain inverted zebrafishes (Rpp), but there was a significant increase in anxiety-like behavior compared with the control group. Which showed consistency to the previous studies. Besides, both non-inverted and wild-type zebrafishes showed a functional dominant on the right-side telencephalon. In contrast, the functional dominant changed to the left-side telencephalon in the brain inverted zebrafish (Rpp). Therefore, it could be inferred that the functional lateralization of the zebrafish would be reversed as the brain inversion.
In conclusion, we suggest the functional lateralization of the telencephalon in the learning and memory does exist in the zebrafish. Further experiments will be required to determine whether the functional lateralization is natural borned (innated) or required the exposure of postnatal experiences (acquired). We also found that even a tiny ablation was performed may have a tremendous impairment effect on both the spatial and emotional learning.

並列關鍵字

zebrafish ； brain lateralization ； anxiety-like behavior ； learning and memory ； parapineal

參考文獻

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