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

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(1)國立台灣師範大學生命科學系博士論文. 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. 研究生: 吳民聰 Ming-Chong Ng 指導教授: 呂國棟博士 Kwok-Tung Lu. 中華民國一○一年七月.

(2) 致. 謝. 本論文得以順利完成，首先要感謝指導教授－呂國棟老師。老師是我生命中的貴人，自大一開始我便在老師實驗室擔任工讀生，十一年的時光轉眼即逝，謝謝老師在師大對我的諄諄教誨與栽培，讓我在專業學識與人格素養上獲益良多。這段亦師亦友的時光歲月，讓我畢生難忘！同時也很感謝嘉大楊奕玲老師、海大呂明偉老師、本系林豊益老師和吳忠信老師撥冗為我口試，提供我許多實驗討論的寶貴意見，以及論文寫作的指導，使本篇論文得以完整呈現。 B211 實驗室就像一個大家庭，雖然大家來自四面八方，但因緣分而讓彼此相聚，這一路走來，大家一同建構了許多難忘的回憶，真的很幸運有這群好夥伴相挺，如果沒有你們的支持與協助，我無法在這段時間裡順利完成此論文，在此容我表達內心的無限感激。古云：長幼有序，一切就遵循古禮吧～感謝明忠助教、伯寬、曜如、孟昌、作豪、郁芳、竣博、世德、于惠、韻莉、惠喻和郁蘭，謝謝你們和我一同渡過研究生涯的每一天，共同用歡笑與汗水編織出生命中的精采樂章。於此特別再次感謝：美食達人曜如，很高興我們能夠共同草創斑馬魚實驗；個性尤如一張白紙的竣博，謝謝你的提醒與督促，讓我實現了人生的夢想；帶我溯溪釣魚的作豪，很高興能夠與你一起學習實驗技巧；聲量控制器壞掉的韻莉，謝謝妳對我魚兒付出的愛心與照顧。以及那些為我研究犧牲的實驗動物們，在此我亦致上最高的敬意與最誠摯的謝意！除了實驗室夥伴，在這離鄉異地求學的日子裡，由衷感謝和我同寢室的好友邦民、政耀、廷翰、哲君和閔楷，謝謝你們平時給予我精神上與生活上的關懷與支持，我會懷念在寢室裡大家彼此相互勉勵、暢談研究夢想的日子…。最後也是最重要的是，感謝家人的關心與照顧，特別是我哥－民耀，每當我遇到生活困境，你總是伸手拉我，帶我走出困境。女友詠晴，謝謝妳對我的包容、關懷和鼓勵，每當我徬徨無助時，妳總是陪著我，使我相信自己的能力，勇敢追求屬於自己的夢想！. I .

(3) 僅以此論文獻給所有幫助我以及關心我的朋友。. 研究生. 僅誌於. 國立台灣師範大學生命科學所民國一○一年七月. II .

(4) Table of Contents Abbreviation Table. ………………………………………………..…….III. List of Figures. ………………………………………………..…….IV. 中文摘要. …………………………………………………..….V. Abstract. ………………………………………………………VII. Chapter 1. .....…………………………………………………………….. 1. Introduction 1. Zebrafish. ……………………………………………………… 2. 2. The telencephalon in ray-finned fishes. ……………………………………… 3. 3. Synaptic plasticity. ……………………………………………………… 5. 4. Fragile X Syndrome. ……………………………………………………… 8. 5. Aim of this study. ……………………………………………………… 14. 6. Figures. ……………………………………………………… 15. Chapter 2. .....…………………………………………………………….. 17. Evoked potentials and synaptic plasticity at the Dl-Dm pathway in slices of the zebrafish telencephalon 1. Introduction. ……………………………………………………… 18. 2. Materials and Methods. ……………………………………………………… 19. 3. Results. ……………………………………………………… 23. 4. Discussion. ……………………………………………………… 28. 5. Figures. ……………………………………………………… 37. III .

(5) Chapter 3. …..…………………………………………………………….. 48. Behavioral and synaptic circuit features in a zebrafish model of Fragile X syndrome. 1. Introduction. ……………………………………………………… 49. 2. Materials and Methods. ……………………………………………………… 50. 3. Results. ……………………………………………………… 58. 4. Discussion. ……………………………………………………… 61. 5. Figures. ……………………………………………………… 66. Chapter 4. .....…………………………………………………………….. 73. Summary and Conclusion. References. .....…………………………………………………………….. 75. IV .

(6) Abbreviation Table DiI. 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate. DHPG. 3,5-dihydroxyphenylglycine. AMPA. α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid. Dm. Dorsal medial. Dl. Dorsal lateral. Dp D. Dorsal posterior. HFS. High-frequency electrical stimulation. LTP. Long-term potentiation. LTD. Long-term depression. LFS. Low-frequency stimulation. MED64. Multi-electrode dish 64-channel. NMDA. N-methyl-d-aspartate. PPF. Paired pulse facilitation. Y. Sulcus ypsiloniformis. TEL. Telencephalon. TTX. Tetrodotoxin. V .

(7) List of Figures. Fig. 1-1………………………………………………………………15 Fig. 1-2………………………………………………………………16 Fig. 2-1………………………………………………………………37 Fig. 2-2………………………………………………………………38 Fig. 2-3………………………………………………………………39 Fig. 2-4………………………………………………………………40 Fig. 2-5………………………………………………………………41 Fig. 2-6………………………………………………………………42 Fig. 2-7………………………………………………………………43 Fig. 2-8………………………………………………………………44 Fig. 2-9……………………………………………………………....45 Fig. 2-10……………………………………………………………..46 Fig. 2-11……………………………………………………………..47 Fig. 3-1………………………………………………………………66 Fig. 3-2………………………………………………………………67 Fig. 3-3………………………………………………………………68 Fig. 3-4………………………………………………………………69 Fig. 3-5………………………………………………………………70 Fig. 3-6………………………………………………………………71 Fig. 3-7………………………………………………………………72. VI .

(8) 中文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 VII .

(9) 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 VIII .

(10) Abstract 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 IX .

(11) 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. X .

(12) Keywords: Zebrafish, Telencephalon, Synaptic plasticity, Mental retardation, Long-term potentiation, Long-term depression.. XI .

(13) CHAPTER 1. Introduction. 1 .

(14) 1. Zebrafish Zebrafish is a small tropical freshwater teleost native to South-East Asia. Zebrafish were first used for biological research purposes by Dr. George Streisinger, a scientist at the University of Oregon, in 1981 (Streisinger et al., 1981, Chen et al., 2005, Souza and Tropepe, 2011). As a relatively simple vertebrate species, the zebrafish are a popular model organism for developmental and genetic studies (Vogel, 2008). Zebrafish are an excellent model for biomedical research, and the advantages of this model include genomic and physiological homology with humans, external fertilization, and large numbers of fertilized eggs. Most importantly, the transparency of the zebrafish embryos allows one to visualize the processes of embryogenesis and morphogenesis in early developmental stages. Moreover, the small size of zebrafish enables cost-efficient maintenance of numerous adult individuals. It is possible to obtain mutant and transgenic strains of zebrafish at a low cost. In 1996, the neuroanatomy of the adult zebrafish brain was described in detail by Rupp et al. (Rupp et al., 1996). Rupp et al. demonstrated that the basic components of the adult zebrafish brain are very similar to those of land vertebrates. Due to the accumulated genetic knowledge and tools developed for the zebrafish, many reports make use of the zebrafish to study complex behaviors such as seizures (Alfaro et al., 2011), addiction (Ninkovic et al., 2006), and learning and memory (Rawashdeh et al., 2007, Blank et al., 2009, Kim et al., 2010, Vuaden et al., 2012). Therefore, zebrafish can be used to help understand the pathways and mechanisms of human neurological disorders and clinical treatments. 2 .

(15) 2. The telencephalon in teleost fishes 2.1. Topography of the Telencephalon Based on neuroanatomical, neurochemical and connectional analysis, the telencephalon of teleost fish is consists of two major subdivisions: the dorsal telencephalic area or pallium, and the ventral telencephlic area of subpallium (Northcutt, 1995, 2006, Mueller et al., 2011). In zebrafish, a lot of the molecular markers for pallial- subpallial boundary have been reports, such as gad67, dlx2a, dlx5a, tbr1 and neurod (Mueller and Guo, 2009, Ganz et al., 2012). Recently, the dorsal telencephalon of zebrafish is further subdivided along the medial-lateral axis into three regions: the dorsal medial (Dm), dorsal lateral (Dl) and the dorsal posterior (Dp) zones, based on the expression of NADPHd and parvalbumin. According to immunohistochemical studies, suggesting the Dm, Dl and Dp are established homologues of the pallial amygdala, hippocampus, and piriform cortex, respectively (Mueller et al., 2011).. 2.2. Connections of the telencephalon In general, the telencephalic connections were investigated by using the fluorescent dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) in fixed brain. The pioneering works of Folgueira has indicated that application of DiI to Dl division led to retrograde labeling Dm division and some labeled fibers crossed the anterior commissure at different rostrocaudal levels, reaching to the contralateral Dl and Dm (Folgueira et al., 2004). Similar results were also reported in 3 .

(16) other teleost fish, such as sturgeon (Huesa et al., 2006) and goldfish (Northcutt, 2006). Thus, histochemical and neuroanatomical evidences suggest that the evolution of telencephalon is highly conserved through phylogenesis, which could have inherited some basic features of brain and behavior organization in teleost fish.. 2.3. The role of telencephalon in memory Convergent lines of evidence indicate that the telencephalon is a critical component of a neural circuit underlying learning and memory in teleost fishes. The pioneering studies revealed that the telencephalon of teleost fish is involved in spatial and emotional learning. For example, whole telencephalon lesions produced a profound conditioned avoidance response deficit in goldfish trained in trace and non-trace procedures (Portavella et al., 2004) . Therefore, behavioral studies suggest that the teleost telencephalon is thought to be involve in information storage and therefore in learning and memory (Savage, 1969, Riedel, 1998). In brief, the telencephalon is functionally homologous to the limbic system in land vertebrates as described by Chandroo et al. (Chandroo et al., 2004). The previous studies on the neural basis of learning and memory in teleost fishes have focused almost exclusively on the role of the dorsal telencephalon, particularly in dorsal lateral (Dl) and the dorsal medial (Dm) division. Increasing experimental evidence suggests that the dorsal pallium of teleost fishes has a predominant role in learning and memory processing (Saito and Watanabe, 2006, Broglio et al., 2010). For example, goldfish with lesions in the Dl region of the telencephalon show 4 .

(17) impairments in reversal spatial learning (Portavella and Vargas, 2005) and allocentric spatial learning (Duran et al., 2008), whereas lesions to the Dm region impair avoidance conditioning (Portavella et al., 2002, Portavella et al., 2004) and spatial learning in an open-field maze (Saito and Watanabe, 2006). In fact, these spatial and emotional deficits in goldfish resemble those observed after damage to the hippocampus and amygdala in amniotes. These data suggest that the Dm and Dl divisions of teleost fish are functionally homologous to the mammalian amygdala and hippocampus, as evidenced by their similar lesion-induced alterations in emotional, spatial learning and memory processing. In this regard, the functional data indicate that the neural basis of learning and memory are remarkably similar between teleost fish and land vertebrate.. 3. Synaptic plasticity Synaptic plasticity can be defined as a modification in synaptic strength and/or response, which is believed to underlie learning and memory processes. A change in the synaptic efficiency that last for periods ranging from milliseconds to a couple of minutes is termed short-term synaptic plasticity whereas a synaptic efficiency lasting for hours, or even weeks is known as long-term plasticity (Alger and Teyler, 1976, Di Filippo et al., 2009, Iezzi et al., 2011).. 3.1. Long-term potentiation (LTP) In 1973, long-term potentiation (LTP) was first discovered in the 5 .

(18) rabbit hippocampus by Tim Bliss and Terje Lamo (Bliss and Lomo, 1973). LTP is an enduring enhancement of synaptic efficacy that has been reported in vitro and in vivo. There are two major forms of LTP that can be classified according to their induction. Most commonly, LTP is induced experimentally by applying brief trains of high-frequency electrical stimulation (HSF) to excitatory synapses. In the CA1 region of the hippocampus, LTP can be induced by HFS via activation of N-methyl-d-aspartate receptors (NMDAR), which leads to calcium influx and, ultimately, sustained postsynaptic intracellular calcium elevation (Collingridge et al., 1983, Lynch et al., 1983, Coan et al., 1987, Calabresi et al., 1992). Several studies have demonstrated that selective competitive antagonists of the NMDAR prevent the induction of the HFS-evoked LTP in hippocampal neurons (Harris et al., 1984, Gozlan et al., 1995, Trommer et al., 1995). Thus, previous studies have revealed that calcium influx through postsynaptic NMDAR is the first step in HFS-induced LTP. Furthermore, a brief application of forskolin (FSK), an adenylyl cyclase activator, can lead to a long-lasting potentiation of synaptic transmission (Lu and Gean, 1999, Broutman and Baudry, 2001, Otmakhov et al., 2004). Most studies have focused on forms of LTP that are triggered by synaptic activation of either NMDARs or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). In NMDAR-LTP, Ca2+ that enters through the NMDARs can activate a variety of protein kinases and/or phosphatases, which in turn moderate synaptic strength. In the NMDAR-LTP model, large increases in intracellular Ca2+ trigger activation of Calcium/Calmodulin-Dependent Protein Kinase II (CaMKII) 6 .

(19) and protein kinase A (PKA) resulting in LTP induction (Abel et al., 1997, Nayak et al., 1998, Duffy et al., 2001, Lee et al., 2009, Sanhueza et al., 2011). In addition, the CaMKII-dependent suppression of protein phosphatase 2A (PP2A) activity and prevention of the dephosphorylation of CaMKII by PP2A might also be involved in the induction and maintenance of LTP (Fukunaga et al., 2000). In the FSK-LTP model, several studies have suggested that FSK triggered AMPA receptor trafficking (Oh et al., 2006, Yudowski et al., 2007) mediates LTP. Over the last few decades, rodent models have been widely used to study the cellular mechanisms of LTP formation. Recently, numerous studies have focused on the electrical activity of specific regions of the adult zebrafish brain, such as the olfactory lobe (Michel and Lubomudrov, 1995, Tabor and Friedrich, 2008), hindbrain (Bliss and Collingridge, 1993, Koyama et al., 2011), and telencephalon (Kim et al., 2004, Nam et al., 2004b). In a previous study, LTP was induced following high potassium treatment in the zebrafish telencephalon (Nam et al., 2004a).. 3.2. Long-term depression (LTD) Long-term depression (LTD) is a reduction in the synaptic strength of neuronal synapses that is involved in learning and memory formation (Ge et al., 2010). There are several types of LTD that can be further classified based on which receptors trigger the process. NMDARs and metabotropic glutamate receptors (mGluRs) are the receptors that most commonly trigger. LTD. (NMDAR-LTD. and. mGluR-LTD,. respectively).. NMDAR-LTD is usually induced by prolonged periods of low-frequency 7 .

(20) stimulation (LFS, 1-5Hz for 5-20 minutes) (Mulkey and Malenka, 1992, Bear and Abraham, 1996, Li et al., 2004). A second major form of LTD, mGluR-LTD, requires the activation of mGluRs, which is induced by the application of the group I mGluR agonist 3,5-dihydroxyphenylglycine (DHPG) (Palmer et al., 1997). Unlike NMDA receptor-dependent LTD, DHPG-induced LTD does not require either Ca2+ influx (Fitzjohn et al., 2001) or serine/threonine protein phosphatase (Kameyama et al., 1998); rather, the activation of protein tyrosine phosphatase is required for the generation of LTD (Moult et al., 2006). Stimulation of NMDA receptors or mGluRs leads to rapid internalization of postsynaptic AMPA receptors, which depresses excitatory transmission and is currently believed to play a critical role in LTD in the hippocampus and cerebellum (Beattie et al., 2000, Carroll et al., 2001, Lee et al., 2002).. 4. Fragile X Syndrome 4.1. Fragile X mental retardation protein (FMRP) The first clinical case of fragile X syndrome was described by James Purdon Martin and Julia Bell in 1943 (Martin and Bell, 1943), but it was not until 1991 that the fragile X mental retardation 1 (fmr1) gene was discovered (Verkerk et al., 1991). Indeed, Fragile X syndrome (FXS) is the most frequent inherited form of human mental retardation, with approximately one in 4000 males and one in 8000 females affected (Turner et al., 1996, Garber et al., 2006). This syndrome is most commonly caused by a triplet repeat expansion (CGG) mutation in the fmr1 gene that is located on the long arm of the X chromosome (xq 27.3) 8 .

(21) (Fu et al., 1991, Oberle et al., 1991, Verkerk et al., 1991). fmr1 polymorphic CGG expansion within the 5´ untranslated region (UTR) of the gene can cause at least two clinically distinct neurological disorders: fragile X-syndrome (FXS), which entails a full mutation (>200 repeats), and fragile X-associated tremor/ataxia syndrome (FXTAS), which entails a premutation (55–200 repeats) (Figure 1-1). In FXS patients, the full-mutation allele leads to hypermethylation (Sutcliffe et al., 1992) and deacetylation (Coffee et al., 1999) of fmr1, which results in the silencing of fmr1 transcription and the absence of the gene product (fragile X mental retardation protein, FMRP) (see Fig 1-1). Although premutation carriers are not at risk for fragile X syndrome, it is an important cause of motor syndromes (tremor or ataxia) in aging FXTAS men (Hagerman and Hagerman, 2004, Baba and Uitti, 2005). FXS is an X-linked inheritable human disease; thus, males are typically more severely affected than females. The clinical features of Fragile X syndrome include physical, developmental and behavioral characteristics. The most prominent physical features of FXS include macroorchidism, an elongated face and a high arched palate that are present in adulthood (Hagerman, 2006, Reiss and Hall, 2007). Clinical studies. have. also. revealed. that. children. with fragile. X. syndrome have behavioral and emotional problems, such as learning disabilities, attention deficit disorder, hyperactivity disorder, anxiety disorder, aggressiveness, hand flapping, and hand biting. Thus, FXS has shown a strong correlation with Autism Spectrum Disorders (ASDs) (Brown et al., 1982, Budimirovic and Kaufmann, 2011). Indeed fmr1 9 .

(22) mutation is the most common genetic cause of ASDs, accounting for approximately 5% of cases (Schaefer and Mendelsohn, 2008). FXS is caused by loss of the fmr1 gene product; the fragile X mental retardation protein (FMRP) is a cytoplasmic mRNA-binding protein that is widely expressed in various tissues with the most abundant expression in the brain and testis (Devys et al., 1993, Hinds et al., 1993). In the brain, the protein is expressed in neurons, particularly those of the hippocampus, amygdala, and in the Purkinje cells of the cerebellum (Abitbol et al., 1993, Devys et al., 1993). FMRP has two KH domains and an RGG domain (RGG box). These domains have been shown to be involved in mRNA binding (Ashley et al., 1993, Siomi et al., 1993a, Siomi et al., 1993b). Amino acid sequence alignment of FMRP from humans, mice, frogs, zebrafish and fruitflies has revealed high conservation in functional domains, including the nuclear localization signal (NLS), two KH domains, the nuclear export signal (NES) and an RGG box (Figure 1-2). However, no CGG repeats have been found in the 5′ UTR sequences of zebrafish, frog or fruit-fly fmr1 gene mRNA (van 't Padje et al., 2005). FMRP plays important roles in the regulation of dendritic mRNA localization and/or synaptic protein synthesis (Feng et al., 1997, Darnell et al., 2001, Zhang et al., 2001, Bassell and Warren, 2008, Kelleher and Bear, 2008). In some case, FXS patients have severe clinical phenotypes that are not caused by the absence of FMRP; rather, they result from mutation in the KH domain. In addition, FMRP has also recently been linked. to. the. microRNA pathway,. an. important. pathway. of. non-coding small RNA sequences (21–23 nucleotides) that regulates gene 10 .

(23) expression (Lee et al., 1993) by modulating associations with Dicer (Cheever and Ceman, 2009). Therefore, recent studies have suggested that dysregulation or loss/dysfunction of FMRP is the cause of FXS-like symptoms.. 4.2. The animal models of Fragile X syndrome 4.2.1. The Drosophila model Previous studies have shown the Drosophila FXS model to be an excellent and simple model system for studying the genetics and molecular aspects of human FXS. Null mutation and overexpression of Drosophila fmr1 (dfmr1) have been widely used to create Drosophila models of FXS (Zhang et al., 2001, Pan et al., 2004). Based on molecular and genetic studies, FMRP function is conserved between Drosophila and humans. For example, Zhang et al. (2001) reported that dFMRP acts as a negative regulator of the microtubule-binding MAP1B homolog Futsch to control synaptic structure and function, suggesting that FMRP acts as a translation repressor in the brain (Zhang et al., 2001). In addition, loss of dFMRP function induced defects in neuronal architecture, disrupted circadian rhythms, impaired social interaction and produced deficits in learning and memory (Dockendorff et al., 2002, Inoue et al., 2002, Morales et al., 2002). The mGluR theory of FXS is the most common mechanistic explanation of the pathophysiology of FXS, as feeding dfmr1 mutants MPEP or class II/III mGluR antagonists can rescue synaptic plasticity, courtship behavior, and mushroom body defects in a Drosophila model of FXS (McBride et al., 2005). Thus, exploring the 11 .

(24) strong evolutionary link between mGluR5 receptors and FMRP signaling in Drosophila may be a useful model for testing the efficacy of therapeutic strategies in FXS.. 4.2.2. The mouse model fmr1 is highly conserved between humans and mice with a coding DNA and amino acid sequence identity of 95% and 97%, respectively; this sequence identity includes the CGG repeat in the 5’ UTR (Ashley et al., 1993). Microscopic analyses of brain material from both FXS patients and fmr1 KO mice have shown dendritic spine abnormalities that include increased spine density and immature spine morphologies in the hippocampus, neocortex, and cerebellum (Hinton et al., 1991, Comery et al., 1997, Nimchinsky et al., 2001, Grossman et al., 2006), suggesting that the loss of FMRP function leads to alterations in dendritic spine structure and deficits of synaptic connectivity and plasticity. fmr1 KO mice exhibit a phenotype with some similarities to humans, such as seizures, macroorchidism and behavioral abnormalities. In behavioral tests, mutant animals displayed hyperactivity, reduced anxiety, reduced social interaction, and learning impairment (Helm et al., 1994, Liu et al., 2011). Consistent with these behavioral deficits, electrophysiological studies have reported the loss of LTP in the anterior cingulate cortex (ACC) and the lateral amygdala (Zhao et al., 2005) and enhanced group I metabotropic glutamate receptor (mGluR)-dependent LTD in the hippocampus (Huber et al., 2002) and cerebellum (Koekkoek et al., 2005) in fmr1 KO mice. 12 .

(25) 4.2.3. The zebrafish model The zebrafish (Danio rerio) is a small tropical freshwater teleost that was first used as a genetic model system in the early 1980s. Due to the accumulated genetic knowledge and tools developed for the zebrafish, they are now considered an excellent and relevant model system in studies of human neurological disorder. Orthologs of the human fmr1 gene have been identified in zebrafish. The deduced amino acid sequence of the zebrafish FMRP consists of 569 residues and shares 72% amino acid identity with human. Pioneering studies found that zebrafish FMRP was ubiquitously expressed throughout embryos at 3 h post-fertilization (hpf); however, in 72-hpf embryos, the most abundant expression of FMRP was in the brain. In adult zebrafish, FMRP is a ubiquitously expressed RNA-binding protein with high expression levels in the telencephalon, diencephalon, metencephalon, spinal cord, cerebellum, and testes (van 't Padje et al., 2005). These findings suggest that zFMRP may play an important role in the developing brain. The zebrafish embryo has been established as a model for fmr1 loss-of-function analysis using a morpholino antisense oligonucleotide to block of FMRP expression (Tucker et al., 2006). These studies suggested that fmr1 and mGluRs have regulatory functions in axonal branching, guidance and fasciculation that have implications for the synaptic morphology component of FXS. However, studies using TILLING (targeted induced local lesions in genomes) to generate fmr1 knockout alleles in zebrafish found that the fish did not display any phenotype; this was in contrast to the morphant reported in a previous study (den Broeder 13 .

(26) et al., 2009). Therefore, it remains to be investigated whether the reported morpholino-based fmr1 phenotypes are due to morpholino’s off-target effects.. 5. Aim of this study For a better understanding of the role of the dorsal telencephalon in learning behavior, it is necessary to unravel the intrinsic circuitry between Dl and Dm divisions, and the functions of the transmitters involved. In the present study, using a multi-electrode array recording system (MED64), the aim of our study was to assess the functional connections between the Dl and Dm regions of a zebrafish telencephalon through analysis of neurotransmission and synaptic plasticity. The loss of- fmr1-function in zebrafish model will be potential to investigate the molecular, pathological and neurobehavioral changes that are common in FXS patients. For a better understanding of the role of the zebrafish FMRP in synaptic plasticity and behavioral performance, we have characterized and compared the electrophysiological characteristics of telencephalon and behavioral tests of wild-type and fmr1 knockout zebrafish. The following issues were attempted in the present study: 1. To characterize the synaptic transmission at Dl-Dm synapses. 2. To examine the synaptic plasticity at Dl-Dm synapses. 3. To identify behavioral deficits in fmr1 KO zebrafish. 4. To investigate whether FMRP plays an important functional role in regulating telencephalic synaptic plasticity in zebrafish 14 .

(27) 6. Figures. Figure 1-1 Schematic of the two distinct pathogenic mechanisms leading to fragile X syndrome and FXTAS. (From Pediatric 123: 378–390, 2009). 15 .

(28) Figure 1-2 The amino acid alignment of Fragile X Mental Retardation 1 (fmr1) proteins (FMRP) shows highly homologous regions of FMRP between human, mouse, frog, fruit-fly and zebrafish. (From Dev. Genes Evol. 215: 198–206, 2005) 16 .

(29) CHAPTER 2. Evoked potentials and synaptic plasticity at the Dl-Dm pathway in slices of the zebrafish telencephalon. 17 .

(30) 1. Introduction The brain is one of the most complex systems in nature, with a structured complex connectivity. The teleostean telencephalon plays an important role in the learning and memory process, particularly, Dl and Dm division involved in spatial and emotional learning. Moreover, a numerous of previous studies have demonstrated the intratelencephalic connections between Dm and Dl are linked by afferent fibers arising from the Dl division.(Huesa et al., 2006, Northcutt, 2006). In this regard, we hypothesized that the intra-telencephalic connection between Dl and Dm may play an important role in the synaptic plasticity of the zebrafish brain. Indeed, there are several types of synaptic plasticity associated with learning and memory, the most important ones known as long-term potentiation (LTP) and long-term depression (LTD) (Maren and Fanselow, 1995, Brigman et al., 2010). In mammals, it is now clear that LTP and LTD have been found to occur in many brain regions involved in learning processes, such as hippocampus, and amygdala. Studies on the CA1 region of hippocampus have demonstrated that LTP and LTD may play in specific types of experience-dependent plasticity. For example, environmental enrichment could restore memory functioning in mice with impaired IL-1 signaling via reinstatement of dentate gyrus LTP (Goshen et al., 2009). Using a multi-electrode array recording system (MED64), the aim of our study was to assess the functional connections between the Dl and Dm regions of a zebrafish telencephalon through analysis of neurotransmission and synaptic plasticity.. 18 .

(31) 2. Materials and methods 2.1. Animals Zebrafish (AB stain) were obtained from the institute of cellular and organismic biology (Academia Sinica, Taiwan) and bred in the vivarium (National Taiwan Normal University). Fish (3 to 5 months of age) of both sexes were used and kept in an aquarium at approximately 26-28 ℃ with a photoperiod of 14-h light/10-h dark. The experimental procedures were approved by the National Taiwan Normal University Animal Care and Utilization Committee (IACUC).. 2.2. Histology Adult zebrafish were sacrificed by euthanasia using ice-cold (0~4℃), artificially oxygenated cerebrospinal fluid (aCSF) solution, fishes head were fixed in PBS, containing. 4% freshly depolymerized para-. formaldehyde at 4°C for 48 h, then the brain was carefully removed from the skull and gently washed in 0.1M PBS (pH 7.3) prior to embedding for sectioning. Fix brains were mounted in 1.5% agarose /15% sucrose and when the agarose was completely solid, place the agarose block into 30% sucrose solution and store at 4°C at least overnight until the block has sunk to the bottom of the tube. Cryosections at 25μm in the coronal plane, and DAPI (blue) was used for nuclear staining.. 19 .

(32) 2.3. Preparation of acute telencephalic slices Fish were euthanized by exposing them to an ice-cold (0~4℃), artificially oxygenated cerebrospinal fluid (aCSF) solution, and their brains were rapidly removed under the aCSF solution. The aCSF contained: 117 mM NaCl, 4.7 mM KCl, 1.2 mM NaH2PO4, 2.5 mM NaHCO3, 1.2 mM MgCl2, 2.5 mM CaCl2, and 11 mM d-(+)-glucose. Subsequently, the whole brain was immersed in a 4% low-melting-point agarose (MDBio, Inc., Taiwan) and transverse telencephalic slices (300 µm) were cut using a vibratome (MA752, Campden Instruments Ltd., UK) and the aid of a microscope. Only one slice was subjected for electrophysiological recording in each animal to minimize the possible rostra-caudal variation. Brain sections were then incubated in an aCSF solution that was bubbled continuously with 95% O2/5% CO2 at least 1 h prior to recordings. They were kept in good condition for at least 4h.. 2.4. Electrophysiological recording Extracellular population spikes were recorded using a 64-channel multi-electrode dish (MED64) system (Alpha MED Sciences, Tokyo, Japan) with a sample rate of 20 kHz. The MED-P515A probe (Alpha MED Sciences, Tokyo, Japan) has 64 microelectrodes arranged in an 8 x 8 grid with an inter-electrode spacing of 150 μm. The size of each electrode is 50 µm x 50 µm. To improve cellular adhesion, before use the surface of the MED64 probe was treated with 0.1% polyethyleneimine (Sigma, St. Louis, MO, USA) in 25 mM borate buffer (pH 8.4) overnight 20 .

(33) at room temperature. For electrophysiological recordings, single brain slices were carefully placed in the recording MED probe and perfused with the aCSF (32℃) at a flow rate of 1-2 ml/min via a peristaltic pump (Gilson Minupuls 3, Villiers Le Bel, France). A nylon mesh and a stainless steel wire were used to secure slice position and contact with electrodes during perfusion. One of the planar microelectrodes, of the 64 available, was used for the stimulating cathode in the dorsal telencephalon. Extracellular field potentials were elicited using bipolar, biphasic rectangular current pulses (0.2 ms duration) applied every 20s via a stimulating electrode placed in the Dl division and population spike were recorded in Dm division. In this study, the maximum population spike response was defined by increasing the stimulus intensity until reaching an asymptotic limit. Thus, the current that produced about 30 to 50% of maximal responses was used throughout the experiment for monitoring test stimulus. Typically, electrophysiological measures of basal synaptic transmission included input-output (I/O) functions and short-term plasticity (paired pulse facilitation, PPF). I/O curves were obtained from fourteen incremental stimulation intensities (20 – 150 μA; bipolar, 0.2 ms duration). The paired pulse ratio (PPR) was determined by calculating the ratio of the average amplitude of the second response to the first. Inter-pulse intervals of the paired pulse stimulation were 20, 50, 100, 150 and 200 ms.. 2.5. Western blot analysis After the animals were killed, the telencephalon brain region was 21 .

(34) quickly removed from the skull and homogenized using a T-PER tissue protein extraction reagent kit (Pierce Biotechnology, Inc., Rockford, IL) with the addition of the Halt Protease Inhibitor Cocktail. The protein concentration was determined by the Bradford protein assay, and an equal amount of protein (25 μg per sample) was subjected to SDS–10% PAGE. The proteins separated on the gel of the SDS–PAGE were transferred to a PVDF membrane (Millipore, Bedford, MA). For the immune-detection, the membrane was first blocked with 5%nonfat milk and 0.05% Tween in PBS for 1 h at room temperature. The primary antibodies used for the detection were rabbit anti-phospho ERK1/2 (1:5,000; Cell Signaling #9101) and anti-GAPDH (1:2,500; Gene Tex, Inc. #GTX82899) antibodies. The membranes were incubated with primary antibodies overnight at 4_C and, subsequently, with HRP-conjugated secondary antibodies for 1 h at room temperature. Finally, the detected signals were visualized with enhanced chemiluminescence (Bioman Scientific Co. Ltd., Taiwan) and quantitatively analyzed by a LAS3000 digital imaging system (Fujifilm, Tokyo, Japan).. 2.6.. Drug application. All drugs were prepared fresh from stock solutions, diluted in aCSF, and applied by superfusion over the slice. Stock solutions of tetrodotoxin (TTX),. 6-cyano-7-nitroquinoxaline-2,3-dione. (CNQX),. and. 2-amino-5-phosphopentanoate (DL-AP5), (R,S)-3,5-Dihydroxyphenylglycine (DHPG, Group I mGlu receptor agonist) were purchased from Ascent Scientific (Bristol, UK) and all made up in distilled water. 22 .

(35) Bicuculline (GABAA receptor antagonist) and forskolin (adenylyl cyclase activator) were also purchased from Ascent Scientific (Bristol, UK) and solved in dimethylsulfoxide (DMSO). DMSO alone (n=3) had no effect on field potentials.. 2.7. Statistical analysis In this study, all values are reported as mean ± SEM. Statistical comparisons of paired pulse facilitation (PPF) was made using the paired t-test. In all cases, p <0.05 was considered to be significant. Statistical analysis was performed using SPSS version 12.0 (SPSS, Chicago).. 3. Results 3.1.. Characterize of Dl-evoked field potentials in the Dm. 3.1.1. Excitatory postsynaptic potentials in Dm division of the telencephalon recorded by multi-electrode arrays (MED64) In Fig. 2-1 an outline of a telencephalon slice border structures is sketched. To recorded evoked field potential, we placed 300 μm thick telencephalon brain over an 8 x 8 MED64 probe with an inter-electrode distance of 150μm (Fig. 2-2A). In 2-2B a bipolar stimulation of Dl division was performed through an electrode located on the border between sulcus ypsiloniformis (Y) and posterior (Dp) zones. Biphasic positive-negative current applied to the stimulation electrode evoked a 23 .

(36) negative peak in Dm. For the first time, the present results reveal that electrical stimulation of the Dl can evoke a negative-going potential in the Dm of zebrafish. Neuronal response properties can be characterized by means of the input-output response function, such as input-output (I-O) curve.. We. characterized. the. input–output. relationship. between. stimulation intensity in the Dl division and the amplitude of population spikes in Dm division. As shown in Fig. 2-3, the amplitude of the population spike gradually rises from threshold values to an approximately linear form, and then smoothly saturates. Our results demonstrating increased excitatory synaptic transmission in response to increased stimulation intensity of Dl afferents.. 3.1.2. Electrical stimulation evoked a field potential in Dm result from direct activation of axons and neurons Electrical stimulation within the dorsal telencephalon evoked a complex field potential, always consisting of an initial positive deflection (P1) followed by a larger negative peak (N2) (Fig. 2-4A). In order to determine the field potential is mediated by synaptic processes, several tests were performed. As illustrated in Fig. 2-4A-C, superfusion of aCSF containing 0.5 mM Ca2+ and 8.0 mM Mg2+ reversibly abolished the N2 component, while P1 was remains intact. From Fig. 2-4C-D it is evident that. 0.5μM TTX abolished all components of the evoked potential.. Moreover, almost all synaptic transmission exhibits either paired-pulse facilitation (PPF), depending mainly on the release probability of terminals. In Fig. 2-5 a typical example of PPF is shown, paired pulse 24 .

(37) stimulation produced a facilitation of the field potential in response to a second stimulus at 20 to 200 ms interpulse intervals (20 ms, 184.5 ± 5%, p<0.01; 50 ms, 176.9 ± 4%, p<0.01; 100 ms, 172.0 ± 7%, p<0.01; 150 ms, 159.8 ± 8%, p<0.01; 200 ms, 146.0 ± 9%, p<0.01). Clearly, the findings indicate that the P1 component of the evoked potential in Dm is a non-synaptic component, N2 a monosynaptic population spike (PS).. 3.1.3 . The transmitter system mediating the synaptic response To investigate whether Dl stimulation is able to activate glutamatergic fibers, slices were exposed to the AMPA/kainate receptor antagonist CNQX. In Fig. 2-6A – C an example of the potent antagonizing action of 5 μM CNQX is shown. It reversibly abolished the amplitudes of population spike, but did not affect the P1 (n=2). Our results suggest that the evoked potential in the Dm of the pallium is mediated through the AMPA/kainate receptor. To test whether NMDA receptors participate in mediating the synaptic response, telencephalon slices were superfused with Mg2+ free aCSF. After perfusion with Mg2+ free aCSF the evoked response was enhanced in amplitude, and a prolonged bursting activity with multiple spike was shown in Fig 2-7B-b. On the other hand, perfusion with the GABAA receptor ( GABAA R) antagonist bicuculline (BIC), which has been shown to be a potent antagonist of inhibitory activity in the telencephalon (Kim et al., 2004). BIC (3 μM) had the same effects, but the duration of prolonged bursting activity with multiple spike was shorter than Mg2+ free aCSF treatment. 25 .

(38) 3.2.. Long-term potentiation at Dl-Dm synapses. 3.2.1. High frequency stimulation (HFS)-induced LTP Long-term potentiation (LTP) has been proposed as a candidate cellular mechanism for learning and memory (Harris et al., 1984). In a previous study, LTP was described in the adult telencephalon brain of the zebrafish (Nam et al., 2004a). However, LTP in subdivisions of dorsal telencephalon remain unclear. Thus, we investigated the expression of LTP in the Dm division induced by high frequency stimulation (HFS) of the Dl division. We showed that the application of three trains HFS sufficient to elicit LTP at the Dl–Dm pathway (Fig. 2-8A), which lasted for at least 1 h. The amplitude of population spike at 1 h after the HFS was 223.4 ± 12% (n = 6, p < 0.01) of baseline (Fig. 2-8B). Previous studies demonstrated that the LTP are NMDA receptor-dependent (Volianskis and Jensen, 2003). This raises a possibility that the glutamatergic NMDA receptor may also be closely related to LTP in the Dl and Dm of the zebrafish. In order to investigate whether NMDA receptors are involved in LTP formation, we examined the effects of an NMDA receptor antagonist, DL-AP5, on the induction phase of LTP. As shown in Fig. 2-9, application of 40 μM of DL-AP5 alone did not produce any significant changes; however, there was a slight reduction in the baseline amplitude of the population spike when compared with control (91.2 ± 6% of the baseline, n = 6, p = 0.168). When LTP-inducing HFS was delivered after incubation with DL-AP5 for 30 min, no significant differences were found in the amplitude of the population spike for up to 30 min after HFS (96.5 ± 5% of the baseline, p = 0.535). 26 .

(39) When the same LTP-inducing HFS was delivered after the washout of DL-AP5, the amplitude of population spike 10 min after HFS were significantly increased (154.8 ± 8% of baseline, p < 0.01), and remained stable for more than 60 min (141.8 ± 8% of baseline, p = 0.019). Thus, our data suggests that NMDA receptor activation is necessary for the induction of LTP by HFS (Fig. 2-9).. 3.2.2. Forskolin (FSK)-induced LTP The activation of adenylyl cyclase results in increased cytosolic levels of cyclic adenosine monophosphate (cAMP) and subsequent action of protein kinase A (PKA). cAMP/PKA transduction cascade has been demonstrated to play a critical role in synaptic plasticity and learning and memory in Drosophila (Davis et al., 1995) and rodents (Vazquez et al., 2000). In present study, we used forskolin, an adenylyl cyclase activator, to examine the effects of cAMP accumulation on synaptic plasticity at the Dl-Dm synapse. As in Figure 2-10A, brief application of forskolin in 50μM for 15 min induced a long-lasting LTP at the Dl–Dm pathway, which lasted for at least 1 h. The amplitude of population spike at 1 h after application of forskolin was 164.0 ± 4% (n = 3, p < 0.01) of baseline. In vehicle experiments, we found that 15 min application of 0.2% DMSO had no effect on basal synaptic transmission, thus the long-lasting effect was elicited by forskolin. In addition, our results demonstrated that the activation of ERK in the telencephalon was induced at 15 min after forskolin (50 μM) bath applied. Only a single protein of 44kDa was clearly detected by phosphor-ERK 1/2 antibodies (Fig 2-10B). 27 .

(40) 3.3 . Long-term depression at Dl-Dm synapses 3.3.1. DHPG-induced LTD To verify that mGluR is important for synaptic plasticity in telencephalon of zebrafish, we examined the effects of application of an mGluR agonist, DHPG, on LTD. We showed that the application of DHPG (25 μM for 10 min) induced LTD at Dl-Dm synapse, which lasted for at least 1 h. The amplitude of population spike at 1 h after the brief application of DHPG was 71.6 ± 13% (n = 6, p < 0.05) of baseline (Fig. 2-11). After 10 min application of 25 μM DHPG, it transient reversibly abolished the amplitudes of population spike, but did not affect the fiber volley. This finding supports our hypothesis that the mGluRs play important roles in synaptic plasticity.. 4. Discussion 4.1. Summary In this study, we will first to identify and characterize synaptic responses. Secondly, the transmitter mechanism mediating the evoked potential will be discussed. Recently, we found that electrical stimulation of the Dl division evoked a negative field potential in the Dm division. The amplitudes of field potential are depending on stimulation intensity. Pairs of stimuli, when delivered at brief inter-pulse intervals (IPI), elicited paired pulse facilitation (PPF). Pharmacological data showed that field potential 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 28 .

(41) 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 GABAergic and glutamatergic transmission may play important roles in the synaptic plasticity of the zebrafish brain. Long-term potentiation (LTP) and long-term depression (LTD) of excitatory synaptic transmission are widespread phenomena expressed at possibly every excitatory synapse in the hippocampus and amygdala. In present study, LTP was induced by high frequency stimulation (HFS) or brief bath application of forskolin. Furthermore, LTD was induced by brief bath application of DHPG. Thus, our results suggest that the intratelencephalic connection between Dl and Dm may play an important role in the synaptic plasticity of the zebrafish brain. It also provides a new electrophysiological. 4.2.. Detailed discussion. 4.2.1. Characterize of Dl-evoked field potentials in the Dm division According to the observations by Northcutt (Northcutt, 2006), tract-tracing studies revealed that neural connections are formed between these regions via afferent Dl fibers projecting to the Dm in goldfish (Cassius auratus). However, the field potential in Dm evoked by Dl stimulation has not been described before. We hypothesized that the connection between Dl and Dm divisions may bear an important intrinsic 29 .

(42) physiological role in synaptic function. In this study, electrophysiological procedures were used to identify and characterize synaptic responses in the Dm region following Dl stimulation in vitro using zebrafish brain preparations. We observed an evoked potential in the Dm division when a single-pulse electrical stimulation was applied to the Dl division, suggesting that the extracellular field potentials are a reliable index of synaptic transmission between the Dl and Dm regions. The field potential can be subdivided into non-synaptic and synaptic components. The following two discuss indicated that P1 component is non-synaptic and N2 is synaptic: first, the amplitude of N2 was invariant during. intensity-response function (I-O. curve). stimulation. Second, superfusion of 0.5 mM Ca. and 2+. paired. pulse. and 8.0 mM Mg. 2+. reversibly abolished N2 but not P1. However, the complete block of field potentials by 0.5 μM TTX showed that all components are dependent on current flow through sodium channels. That evidence suggests that P1 component consists of extracellular spikes not generated through synapses. Herein, we suggesting that the P1 component can be identified as a fiber volley (FV), because Henze et al. (1997) observed that the electrical stimulation of the hippocampal mossy fibers (MF) results in evoked a positive deflection fiber volley (FV) waveform in area CA3 (Henze et al., 1997). Data from present study showed that N2 component as a population spike (PS) by the electrical stimulation of Dl division. In addition, data showing that the onset latency of PS is slightly decreased upon increasing stimulus amplitude (see Figure 2-3 and Figure 2-7A), suggesting that the 30 .

(43) PS is generated through monosynaptic pathway, which consistent with previous studies (Pennartz et al., 1990, Henze et al., 1997, Lewis and O'Donnell, 2000). In attempting to identify the transmitter system mediating the Dl evoked synaptic response, the following results are of important: (1) superfusion of Mg2+ free aCSF or application of the GABAA antagonist bicuculline (BIC) significantly enhanced the population spike. (2) 5 μM CNQX, an AMPA receptor antagonist completely abolished the population spike, but did not affected the volley fiber. In neuron, most of the excitatory synaptic signaling is mediated by glutamatergic. neurotransmission.. Glutamate. receptors. can be divided into two major classes: non- NMDA and NMDA receptors. Non-NMDA receptors, such as AMPA and kainite receptor, mediate rapid excitatory synaptic transmission while NMDA receptors play important roles in neuronal plasticity and development. In zebrafish, glutamatergic neurotransmission has been accumulated in numerous areas of the brain, such as retina (Yazulla and Studholme, 2001) and olfactory bud (Edwards and Michel, 2002, 2003). In this study, the Dm field potential was reversibly blocked by CNQX and enhanced by superfusion of Mg2+ free aCSF, suggesting that Dl-Dm pathway synaptic responses are mediated by the glutamatergic neurotransmission. Collectively, these findings are in line with previous studies that have described the field potential in the posterior telencephalon of the zebrafish (Nam et al., 2004a). The amino acid γ-aminobutyric acid (GABA) represents one of major inhibitory neurotransmitters in the mammalian central nervous system 31 .

(44) that has historically received the most attention in alcohol research. In neuron, GABA is synthesized from glutamic acid by glutamic acid decarboxylase (GAD). Recently, zebrafish has been proposed as a models organism for human disorder diseases, such as alcoholism and seizure. Therefore, information on the function role of GABA neurotransmitter system in zebrafish brain is becoming valuable. Here, we found that the GABAA receptor-mediated inhibition occurs in Dl-Dm synaptic circuit. Consistent with previous study, in which induced an epileptiform burst in population spikes recorded in the posterior dorsal telencephalon (Kim et al., 2004). We suggest that telencephalic GABAergic system may involve in epileptic seizure activity in zebrafish.. 4.2.2. Long-term potentiation at Dl-Dm synapses In present study, electrophysiological procedures were used to identify and characterize LTP in the Dm region following Dl stimulation in vitro using zebrafish brain preparations. We show that the tetanization of the Dl region induces a robust and stable NMDA receptor-dependent LTP at Dm division. The N-Methyl-D-aspartate (NMDA) receptor is a special type of ionotropic glutamate receptor. It is widely distributed in mammalian brain areas that are implicated in learning and memory, such as the hippocampus and amygdala. In neurons, the activation of the NMDA receptor stimulates Ca2+ influx, leading to the elevation of postsynaptic Ca2+ levels and the subsequent initiation of certain intracellular signal transduction cascades. The NMDA receptor also plays an important role in synaptic plasticity and learning processes. As 32 .

(45) reported in previous studies, the high frequency stimulation of the NMDA receptor could evoke long-term potentiation (LTP) in hippocampal slices, which appeared to be a cellular mechanism underlying synaptic plasticity and, ultimately, learning and memory (Bliss and Collingridge, 1993, Beck et al., 2000). The involvement of the NMDA receptor in cognitive function has been further demonstrated in rodents on the basis of their behavioral consequences following the inactivation of the NMDA receptor using specific antagonists. The intrahippocampal infusion of an NMDA receptor antagonist, AP5, has been demonstrated to cause a significant impairment in the inhibitory avoidance task (Izquierdo et al., 1992, Roesler et al., 1998). In teleost fish, the telencephalic NMDA receptor plays an important role in LTP formation (Nam et al., 2004b) and in spatial (Gomez et al., 2006) and avoidance learning (Xu et al., 2003). In this study, we found that LTP induction was blocked during a period of DL-AP5 perfusion. However, upon washout of DL-AP5, LTP could be induced. This suggests that an NMDA receptor-mediated component to long-term synaptic plasticity in the Dl–Dm synapses, that may be involved in regulating learning and memory processes. The extracellular signal-regulated kinase (ERK) pathway is a vital cascade initiated by the stimulation of the NMDA receptor. NMDA receptor signal transduction is implicated in diverse cellular processes, including cell growth, proliferation, differentiation (Marshall, 1995, Ballif and Blenis, 2001), and learning and memory (Quevedo et al., 2004). Consequently, it is not surprising to find that the physiological 33 .

(46) biochemical roles of ERK are highly conserved among vertebrates (Robinson and Cobb, 1997). For example, the FGF/ERK signaling process has been shown to be involved in the induction and patterning of the telencephalon in both mice and zebrafish (Shinya et al., 2001). Recent studies also have shown that the NMDA receptor-modulated ERK activation is required for fear conditioning in the zebrafish. These findings demonstrate that the ERK signal cascade in the telencephalon of zebrafish may also be closely related to LTP. As was expected, in the present study, the activation of ERK in the telencephalon was induced at 20 min after application of forskolin (50μM). This is consistent with the finding that activation of PKA by application of forskolin to hippocampal slices results in ERK activation in CA1 region (Kanterewicz et al., 2000). In the present study, we confirmed that cAMP signaling pathway is also required for induction of LTP at Dl-Dm synapse.. 4.2.3. Long-term depression at Dl-Dm synapses We show that pharmacological activation of mGluR5 by application of DHPG could triggers a robust, stable and long-lasting LTD. This is consistent with previous observations that DHPG-induced LTD in hippocampal CA1 synapses (Palmer et al., 1997, Fitzjohn et al., 1999, Schnabel et al., 2001, Tokay et al., 2009). The mGluR5 is constitutively expressed and regulates neuronal ion channel activity such as AMPA receptor. In present study, after 10 min application of 25μM DHPG, the field potential response was significantly reduced and continued to 34 .

(47) decline until completely abolished. A recent study showed that DHPG induced depression was associated with the internalization of AMPA receptors, which results in removal of AMPA receptors from the synapse (Xiao. et. al.,. 2001).. Therefore,. here. we. have. shown. that. adult zebrafish and mammalian synapses may share a common molecular mechanism that regulates LTD induction.. 4.3. Physiological significance According to the observations by Maren and Fanselow (Maren and Fanselow, 1995), the axonal projections from the hippocampus to the amygdala might play an important role in synaptic plasticity and context conditioning in the rat. Currently, the telencephalon region of teleost fish has been revealed as an important component of learning and memory, including spatial memory (Saito and Watanabe, 2006, Duran et al., 2008, Broglio et al., 2010) and emotional memory (Portavella et al., 2004, Portavella and Vargas, 2005). In addition, anatomical and functional studies suggest that the Dm and Dl divisions of the telencephalon are homologues of the mammalian amygdala and hippocampus, respectively (Braford, 1995, Mueller et al., 2011). Thus, identification of the teleost telencephalic synaptic circuit is of great interest since telencephalons is playing a key role in many neurological conditions. In summary, our data shows that extracellular field potentials can be evoked in the Dm division following Dl stimulation in vitro. More specifically, the Dm region exhibited both short- and long-term forms of synaptic plasticity, as well as paired pulse facilitation (PPF), long-term potentiation (LTP) and 35 .

(48) long-term depression (LTD). These studies have emphasized the possible neural interaction between the Dl and Dm regions in dorsal telencephalon. Here, we provide the first evidence that suggests the projection from Dl to Dm divisions in zebrafish could correspond to the similar projection from the hippocampus to amygdala in rodents. Our findings present a new possibility for the role of neural connections between the Dl and Dm regions in the mechanisms of learning and memory. Taken together, this knowledge will facilitate use of zebrafish as a model for neurobiological research and as a model for human neurological disorder.. 36 .

(49) 5. Figures. Figure 2-1 Lateral (A), dorsal (B) and coronal plane (C) views of the adult zebrafish brain. The box shows the coronal section in the telencephalon as illustrated in (C). The dorsal telencephalon of zebrafish is further. subdivided along the medial-lateral axis into three regions: the dorsal medial (Dm), dorsal lateral (Dl) and the dorsal posterior (Dp) zones. The sulcus ypsiloniformis (Y) appears as a small indentation between the Dm and Dl zone.. 37 .

(50) Figure 2-2 The field potentials evoked in the dorsal pallium by stimulation of the lateral division (Dl) of the pallium. (A) The telencephalon was placed over the electrode of an MED probe (single electrode size: 50 μm; inter-electrode distance: 150 μm) and suitable electrodes (white square) were selected for the stimulating cathode and recording cathode (white open square). The sulcus ypsiloniformis (Y) marked the border between the Dm and Dl divisions. (B) Representative network traces of field potential recorded across the 16 sites following electrical test stimulation through one electrode #29 on the telencephalon slice. The numerical values indicate the number of electrode aligned on the MED64 probe from 1 to 64 (8×8 array).. 38 .

(51) Figure 2-3 The input-output relationship at the Dl-Dm pathway of telencephalon slices. Mean input-output curve was generated by plotting population spike amplitude (mV) over across a range of input stimulus intensities (μA). Population spike amplitude was calculated as the mean of the amplitude of the two consecutive positive peaks to the maximal negative peak.. 39 .

(52) Figure 2-4 The P1 components of the locally evoked field potential result from direct activation of axons and neurons, whereas N2 are synaptically mediated. In A, the components of the field potential are indicated. A-C: N2 was reversibly blocked by application of 0.5 mM Ca. 2+. and 8.0 mM Mg. 2+. perfusion, while P1 was not affected. C, D: all components of the field potential was abolished by 0.5 μM TTX.. 40 .

(53) Figure 2-5 Paired pulse facilitation of population spike recordings in the dorsal telencephalon. (A) Following paired pulse stimulation of the Dl division, extracellular population spike in the Dm division were collected using varying interpulse intervals (20, 50, 100, 150, 200 ms). (B) The plot summarizes facilitation of the second potential relative to the first one as a function of the short intervals (<200 ms). The amplitude of the population spike were analyzed and all values are reported as the mean ± SEM from 9 slices.. ＊＊. p < 0.01.. 41 .

(54) Figure 2-6 AMPA/kainate receptor antagonists reversibly abolish the synaptic response to Dl stimulation. In this experiment, 5 μM CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) reversibly abolished the field potential but did not affect the CAP. Washout time was about 60 minutes for CNQX.. 42 .

(55) Figure 2-7 Effects of Mg2+ free aCSF and bicuculline (BIC) bath application on population spike from telencephalon slices. (A) Overlay of population spike responses at increasing stimulation intensities. In the following experiment, the Test stimulus intensity was adjusted to a level that produced a population spike of 50% of the maximum response. (B) Mg2+ free aCSF produces an epileptiform burst in population spike recorded from Dm. a: single population spike was induced in normal aCSF. b: the evoked bursting activity during perfusion with Mg2+-free aCSF. c: these effects were reversible after wash with normal aCSF. (C) BIC enhances the population spike and produces an epileptiform burst in population spike recorded from Dm. a: single population spike was induced in normal aCSF. b: the amplitude of the population spike was enhanced and the epileptiform activity appeared following application of 3μM BIC. c: these effects were partially recovery after 2 hr wash. 43 .

(56) Figure 2-8 LTP was induced by application of high frequency stimulation (HFS, arrows) in the dorsal telencephalon. (A) A single experiment, the LTP induced by three trains of HFS treatment in the Dl division. LTP-inducing HFS produced a robust and lasting potentiation for 120 min. (B) The average of experiments from 6 slices was used. Each point represents the mean ± SEM of the population spike amplitude. The waveform, recorded before tetanization (solid line) and 1 h post-tetanus (dashed line).. 44 .

(57) Figure 2-9 The effect of NMDA receptor antagonist, DL-AP5 on LTP induction. After 15 min of baseline recording, slices were perfused with 40 μM of DL-AP5 (solid line) for 30 min. The first HFS (arrows) was delivered 20 min after the start of the drug per-fusion. The 2nd HFS was delivered 30 min after the washout the DL-AP5. After HFS, stimulation and recording were paused for 10 min for stabilization of the evoked response. LTP induction was blocked when the first HFS was delivered during the period of AP5 superfusion, but not by 2nd HFS after DL-AP5 was washed out.. 45 .

(58) Figure 2-10 Long-lasting potentiation of field potential induced by forskolin. (A) After 15 min of baseline recording, slices were perfused with 50μM of forskolin for 15 min. The application of forskolin induced LTP that lasted for at least for 60 min. The application of vehicle alone (0.2% DMSO) had no effect on synaptic transmission. The sample traces taken from experiment at the times indicated. (B) Representative Western blots showing regulation of phosphorylated ERK (P-ERK) levels in telencephalon by forskolin. 46 .

(59) Figure 2-11 Brief application of the mGluR agonist DHPG induced LTD at Dl-Dm synapses. After application of 25μM DHPG for 10 min, the PS amplitude was substantially depressed and DHPG leads to a long-lasting depression of synaptic transmission that lasted for at least for 1 h.. 47 .

(60) CHAPTER 3. Behavioral and synaptic circuit features in a zebrafish model of Fragile X syndrome.. 48 .