應用斑馬魚作為研究端腦突觸可塑性及智能障礙疾病的模式

The zebrafish as a model for studying of telencephalic synaptic plasticity and mental retardation

吳民聰(Ming-Chong Ng)

指導教授 ： 呂國棟

國立臺灣師範大學/理學院/生命科學研究所/博士(2012年)

摘要

硬骨魚類的端腦在學習與記憶的形成過程中扮演著重要的角色，其中又以端腦背側的外側區(Dl)與中側區(Dm)最為關鍵。利用螢光追蹤方法可發現，將螢光染劑置入D1區後，螢光物質會由Dl往Dm傳遞，這現象意味著兩者之間的神經纖維有緊密相連的關係，但目前探討Dl－Dm間突觸傳遞現象的研究還非常稀少。斑馬魚是一種廣泛應用於探討藥物成癮、焦慮以及學習和記憶等研究的模式動物。本論文的研究目的之一即以電生理技術，探討在斑馬魚端腦中Dl－Dm投射路徑的神經傳遞與突觸可塑(synaptic plasticity)現象。從結果可觀察到，在Dl給予一次電刺激能引發Dm產生一個負電位之電場電位(field potential, FP)，且該FP能被AMPA/kainate受器拮抗劑CNQX、0.5 mM Ca 2+、8.0 mM Mg 2+ 及TTX (0.5 μM)所阻斷；相反的，在無Mg 2+的人工腦脊髓液以及bicuculline中FP則能被提升並引發神經的猝發(bursting)現象。以上結果意味著興奮性與抑制性的神經傳遞作用皆可能具調節神經突觸的功能。為了探究這假說，本論文進一步探討了突觸可塑現象中的長期增益效應(LTP)與長期抑制效應(LTD) 。由結果發現，連續三次高頻刺激(每秒100Hz)或投予腺苷酸環化酶啟動劑Forskolin (50 μM) 15分鐘後皆可引發LTP現象，前者為NMDA受器依賴性LTP，而後者需要extracellular related-signal kinase (ERK)的參與。此外，投予代謝型谷氨酸受體興奮劑DHPG　(25 μM)　10分鐘後，則會引發持續至少１小時的LTD現象 。由此可知，斑馬魚端腦Dl與Dm間的突觸連結為端腦突觸可塑性的關鍵角色，也在探討斑馬魚學習與記憶之神經機轉上提供了一個新的電生理模式。另外，斑馬魚在發生遺傳學等相關人類疾病的研究中也已成為不可或缺的動物模式。X染色體脆折症(Fragile X syndrome, FXS)是發生率較高的人類遺傳性智能遲滯疾病，伴隨著外型異常、認知功能以及行為障礙等症狀。FXS是由於FMR1基因發生突變造成其蛋白FMRP缺失所致，建立FXS的動物模式將有助於我們進一步瞭解致病的細胞與分子機制。因此，本論文的另一研究目的即為利用FMR1基因缺失斑馬魚，探究FMRP在行為及神經突觸可塑性中所扮演的角色。實驗結果顯示，成年斑馬魚因缺乏FMR1基因表達，而產生低焦慮、過動和抑制性逃避性學習障礙現象。而在電生理上，FMRP的缺失對於突觸傳遞功能並無明顯影響，但在突觸可塑性方面，相較於對照組，FMR1剔除斑馬魚端腦LTP的強度會減弱，相反的LTD則增強。綜合此研究的各項重要發現，我們認為FMR1基因剔除斑馬魚在未來應用上，除有助於我們瞭解FXS的致病機轉外，更能協助治療性藥物的開發。

關鍵字

斑馬魚 ； 端腦 ； 突觸可塑性 ； 智能障礙 ； 長期增益效應 ； 長期抑制效應

並列摘要

In teleost fishes, the lateral (Dl) and medial (Dm) division of the dorsal telencephalon are important in learning and memory formation. Tract-tracing studies revealed that neural connections are formed between these regions via afferent Dl fibers projecting to the Dm. However, research analyzing Dl–Dm synaptic transmission is scant. Ray-finned zebrafish has been a widely used model organism in behavioral research such as addiction, anxiety, and in learning and memory. Purpose of present dissertation was to investigate neurotransmission and synaptic plasticity in projections from the Dl to the Dm in zebrafish using electrophysiological techniques. The results demonstrated that electrical stimulation of the Dl division evoked a negative field potential (FP) in the Dm division. In addition, pharmacological data showed that FP in the Dm division could be inhibited by application of the AMPA/kainate receptor antagonist, CNQX (5μM), 0.5 mM Ca 2+ and 8.0 mM Mg 2+ and TTX (0.5 μM). In contrast, Mg2+ free aCSF and bicuculline upon synaptic responses and prolonged bursting activity with multiple spikes in the Dm division. These results suggest that both glutamatergic and GABAergic transmission play a role in modulation of synaptic function. To test this hypothesis, we analyzed two major forms of synaptic plasticity, long-term potentiation (LTP) and long-term depression (LTD). In this study, NMDAR-dependent LTP, induced through the application of three trains of high frequency stimulation (HFS; 100 Hz for 1 s). Moreover, a brief application of Forskolin (50μM, 15 min), an adenylyl cyclase activator, can lead to a long-lasting potentiation of synaptic transmission via activation of extracellular related-signal kinase (ERK). LTD is opposite effect to LTP, the application of DHPG, group I mGluR agonist (25 μM for 10 min) induced LTD, which lasted for at least 1 h. Our results suggest that the intratelencephalic connection between Dl and Dm may play an important role in the synaptic plasticity of the zebrafish telencephalon. It also provides a new electrophysiological model for studying the neural mechanisms underlying learning and memory in zebrafish. Fragile X syndrome (FXS), the most frequent inherited form of human mental retardation characterized by the physical, cognitive impairment and behavioral problems, is caused by silencing of fmr1 transcription, and absence of the FMR1 protein (FMRP). Recently, the animals models of FXS have been greatly facilitated the investigation of molecular and cellular mechanism of this loss-of-function disorder. The present study is aimed to further characterize the role of FMRP in behavior and synaptic function by using fmr1 knockout zebrafish. On adult zebrafish, we found that fmr1 knockout animals to produce anxiolytic-like responses with increased exploratory behavior in light/dark and open-field tests, and avoidance learning impairment. Furthermore, electrophysiological recordings from telencephalic slice preparation of knockout fishes displayed markedly reduced long-term potential and enhanced long-term depression as compared to wild-type fishes, however, basal glutamatergic transmission and presynaptic function at the Dl-Dm synapse was remains normal. Taken together, our study suggests that zebrafish has valuable potential as a complementary vertebrate model to study the molecular pathogenesis of the FXS.

並列關鍵字

Zebrafish ； Telencephlaon ； Synaptic plasticity ； Mental retardation ； Long-term potentiation ； Long-term depression

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