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

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(1)國⽴立臺灣師範⼤大學⽣生命科學系碩⼠士論⽂文. 利⽤用離體電⽣生理模式探討斑⾺馬⿂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. 研究⽣生：. 陳郁嵐 Yu-Lan Chen. 指導教授：呂國棟博⼠士 Kwok-Tung Lu. 中華民國 105 年 2 ⽉月.

(2) Table of Contents Abbreviation Table .....................................................................................3 中文摘要 ....................................................................................................5 Abstract ......................................................................................................7 1. Introduction ..........................................................................................10 1.1. Zebrafish ...................................................................................10 1.2. Cerebral lateralization ...............................................................12 1.3. Teleost fish telencephalon.........................................................14 1.3.1 Anatomical structure of telencephalon .....................................14 1.3.2 Connections of telencephalon ...................................................15 1.3.3 The role of telencephalon in learning and memory ..................16 1.4. Synaptic plasticity ......................................................................17 1.4.1 Long-term potentiation (LTP) ..................................................17 1.4.2 Long-term depression (LTD) ...................................................19 2. Research aim ........................................................................................21 3. Materials and methods .........................................................................22 3.1. Experimental animals .................................................................22 3.2. Brain slice preparation ................................................................22 3.3. Electrophysiological recording ...................................................23 3.4. Drug application .........................................................................24 3.5. Real-time polymerase chain reaction (q-PCR) ...........................24 RNA extraction ..................................................................................25 RNA reverse transcription .................................................................25 3.6. Statistical analysis ......................................................................28 4. Results ..................................................................................................29 4.1. Excitatory postsynaptic potentials in Dm division of the telencephalon recorded by multi-electrode arrays (MED64) ............29 4.2. Dl-evoked long-term potentiation (LTP) in Dm division of contralateral side ................................................................................30 High frequency stimulation (HFS)-induced LTP ..............................30 4.3. Dl-evoked long-term depression (LTD) in Dm division of contralateral side ................................................................................36 4.3.1. Low frequency stimulation (LFS)-induced LTD ....................36 4.3.2. DHPG-induced long-term depression .....................................38 . 1 .

(3) 4.4. Characteristics of field potentials of Dl-evoked field potentials in Dm division of contralateral side ......................................................40 Excitatory postsynaptic potentials in Dm division of contralateral side of the telencephalon ...................................................................40 4.5. NMDA receptor distribution of left and right hemisphere of telencephalon .....................................................................................43 5. Discussion ............................................................................................44 6. Reference ..............................................................................................52 7. Figures ..................................................................................................62. . . 2 .

(4) Abbreviation Table AMPA. α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid. AP5. 2-amino-5-phosphonopentanoic acid. aCSF. artificial cerebrospinal fluid. CaMK. calcium/calmodulin-dependent protein kinase Ⅱ. DiI. 1, 1'-dioctadecyl-3, 3, 3’, 3’-tetramethylindocarbocyanine perchlorate. DHPG. 3,5-dihydroxyphenylglycine. Dm. dorsal medial. Dl. dorsal lateral. Dp. dorsal posterior. FV. fiber volley. FSK. forskolin. HFS. high frequency electrical stimulation. LFS. low frequency electrical stimulation. LTP. long-term potentiation. LTD. long-term depression. LES. left eye system. LY367385. (S)-(+)-a-amino-4-carboxy-2-methylbenzeneacetic acid. MED64. multi-electrode dish 64-channel. mGluRs. metabotropic glutamate receptors. NMDA. N-methyl-D-aspartate. NADPHd. nicotine adenine dinucleotide phosphate diaphorase. . 3 .

(5) PP2A. protein phosphatase 2A. PPF. pair pulse facilitation. PKA. protein kinase A. q-PCR. real-time polymerase chain reaction. RES. right eye system. TEL. telencephalon. TTX. tetrodotoxin. . . 4 .

(6) 中文摘要腦側化(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 . 5 .

(7) 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。關鍵字：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 . 6 .

(8) Abstract 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 . 7 .

(9) 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) . 8 .

(10) 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.. Key words: cerebral lateralization, contralateral, electrophysiological techniques, ipsilateral, long-term depression, long-term potentiation, NMDA receptor, specialization, synaptic plasticity, telencephalon, zebrafish . 9 .

(11) 1. Introduction Research background 1.1. Zebrafish Zebrafish is a tropical freshwater teleost belonging to family Cyprinidae of the order Cypriniformes. Being native inhabited to the southeastern stream of Himalayan region. Its body color is silver gray, covered with dark blue stripes and an adult zebrafish can grow to 4 centimeters in length. Zebrafish was first introduced as scientific research purpose in 1981 by the American biologist, Dr. George Streisinger in University of Oregon, investigating neural development of vertebrates on genetic level (Streisinger et al 1981). Zebrafish exhibits the features of vertebrates from simplicity to complexity on embryo development, neural development, and development of immune system (Guo 2004, Sertori et al 2015, Tabassum et al 2015). As a biomedical research animal model, zebrafish possesses numerous advantages, including being diploid, external fertilization and hundreds of eggs in each clutch, cost-efficient maintenance and being highly genomic and physiologically resemble human. Most important of all, the transparent zebrafish embryo benefits the study of morphogenesis and embryogenesis process in early developmental stage (Vogel 2008, Yang et al 2014). Zebrafish is highly genetically conserved, and homologous with mammals (Cerda et al 1998, Barriga et al 2015). Therefore, thousands of transgenic strains have been generated using genetic engineering techniques. Different strains vary in . 10 .

(12) organ development and differentiation, physiological response and behavior performance. Various mutant and transgenic strains of zebrafish are ideal materials for developmental studies. (Wittbrodt & Rosa 1994, Kikuchi et al 1997, Roosing et al 2014), such as genetic manipulation on gene of fragile X mental retardation 1 (fmr1) produces zebrafish with fragile X syndrome and serves as a model for human hereditary retardation (Ng et al 2013, Kim et al 2014, Shamay-Ramot et al 2015). Rodent models were frequently used for investigating developmental related genes and/or outcomes of embryonic development. With the advantages of well-defined developmental genes and cost-efficient maintenance, zebrafish has become popular model for developmental and genetic studies in recent years. In addition, Food and Drug Administration (FDA) formally included zebrafish as preclinical study animal model in 2013. Due to the maturation of experimental techniques used in zebrafish studies and understanding of zebrafish on genetics, it is also a suitable model for complicated behavioral studies, such as drug addiction (Gerlai et al 2000, Darland & Dowling 2001), learning and memory (Zala & Maattanen 2013, Luchiari et al 2015), epilepsy (Cunliffe 2015, Dinday & Baraban 2015) and so forth. Zebrafish shows spontaneous muscle contraction 41 hours after fertilization, using a simple spinal network consists of sensory neuron, interneuron and motor neuron (Saint-Amant & Drapeau 2001). 51 hours after fertilization, zebrafish shows a reflective . 11 .

(13) short swimming distance response towards slight stimulation. 5 days after fertilization, zebrafish shows autonomous behavior in response to stimulation, such as escape response towards the source of impact, or goal-directed response and escape response towards food and predator respectively. It also shows left side or right side optomotor response following different kinds of visual stimulation. Not only adult zebrafish can be used in behavioral studies, also juvenile fish as well. A study conducted by Rupp et al. in 1996, they made a detailed description of neural anatomical structure of adult zebrafish (Rupp et al 1996). According to their results, the brain structure of an adult zebrafish is similar to those of land vertebrates. Therefore, to identify the homologous brain area of land vertebrates and teleost was easier than those of invertebrates. Previous studies indicate that limbic system, especially hippocampus, is responsible for cognition, memory assessment and formation for land vertebrates (Rawlins et al 1993, Alvarez & Ruarte 2002). The functions of telencephalon are similar to those of hippocampus (Rodriguez et al 2005, Salas et al 2006). Thus, zebrafish has been used as a model to understand mechanisms of human neurological disorders and clinical treatments (Biava et al 2015, Lepesant 2015).. 1.2. Cerebral lateralization Cerebral lateralization, also known as functional lateralization, refers to two cerebral hemispheres that have its own specialized function. . 12 .

(14) Division of either side of the brain could be responsible for a particular function of the contralateral side. Different brain area of Left and right cerebral hemispheres is responsible for different function, being complimentary to one another without conflict, and make an individual more coordinate on cognitive behavioral performance (Dadda et al 2009, Gotts et al 2013), and then the performance of social behavior (Dadda et al 2010). Take mammals for example, the structures of left and right cerebral hemisphere are different, controlling different area and function. For instance, handedness, also knew as dominant hand in human. About 90% of the population is right-handed, and their left cortical cortex is responsible for functions related to analysis, logic, mathematics, and languages; the right cortical cortex manages functions related to emotion, creativity, art and spatial perception. The phenomenon of functional lateralization was observed not only in the brain of mammals, but also in organisms with bilateral nervous system such as amphibians, reptiles, birds and fishes (Bisazza et al 1998, Stancher et al 2006, Bonati et al 2008), for it is highly conserved on evolution. Early studies on functional lateralization used chickens born four to five days as animal model and placing food in the sight of chickens as attraction. If the food was placed at right sight of chicken, it will peck the food directly without hesitation, and if the food was placed at left sight of chicken, it will not peck before a period of observation. The results suggested that stimulus was perceived by different eye depending on their position, and then delivered . 13 .

(15) to distinct hemisphere of brain. At last, resulting in different response (Andrew et al 2000). Because zebrafish has the advantages in genetic and developmental studies (Ingham 1997), it has been used to conduct studies related to functional lateralization in recent years. Previous studies suggested that no matter adult zebrafish or juvenile zebrafish, it showed eye use preference depending on different types of stimulus, and the phenomenon was called left eye system (LES) and right eye system (RES). In general, zebrafish tends to use right eye to observe familiar objects or food, and uses left eye to observe unfamiliar environment and objects for risk assessment (Miklosi & Andrew 1999, Sovrano 2004, Watkins et al 2004, Sovrano & Andrew 2006, Andersson et al 2015). Researchers used different attributes of stimulus to observe the behavioral response in zebrafish, and they proved zebrafish indeed showed selectivity on eye use preference. It served as an evidence of cerebral lateralization of zebrafish.. 1.3. Teleost fish telencephalon 1.3.1 Anatomical structure of telencephalon The telencephalon of teleost is divides into two major areas according to neurochemical staining methods and neuroanatomical analysis. The two major areas are dorsal telencephalic area of pallium and ventral telencephalic area of subpallium. In zebrafish, there are lots of existing molecular markers for pallial-subpallial boundary, such as dlx2a、︑､ . 14 .

(16) dlx5a、︑､tbr1、︑､gad67 and neurod (Mueller & Guo 2009, Mueller & Wullimann 2009, Ganz et al 2012). Further studies discovered that dorsal telencephalon of zebrafish divided into three regions along the medial-lateral axis: the dorsal lateral (Dl), dorsal medial (Dm) and dorsal posterior (Dp) based on anatomical expression of parvalbumin and nicotine adenine dinucleotide phosphate diaphorase (NADPHd) and in subsequent BrdU labeling experiments, evidence of labeled migrating cells indicated the Dl, Dm and Dp region of zebrafish are highly homologous to hippocampus, amygdala and piriform cortex respectively (Mueller et al 2011).. 1.3.2 Connections of telencephalon The early studies conducted by Folgueira et al. indicated the connection of telencephalon by using the fluorescent dye DiI (1,1’-dioctadecyl-3, 3, 3’, 3’-tetramethylindocarbocyanine perchlorate) in fixed brain. By applying DiI to the Dl division, DiI labeled Dm division, the fibers at different rostrocaudal levels crossed the anterior commissure and Dl, Dm of the contralateral side. The character of telencephalon connection has been observed in other teleost, such as goldfish (Villani et al 1996) and sturgeon (Folgueira et al 2004). Neuroanatomical and histochemical evidence suggested that telencephalon is an evolutionary highly conserved brain region through phylogenesis.. . 15 .

(17) 1.3.3 The role of telencephalon in learning and memory Previous studies of convergent line indicated that telencephalon is a crucial composition of neural circuit forming learning and memory in teleost fish (Salas et al 2006). Studies revealed that telencephalon is also related to formation of emotion and spatial learning (Portavella et al 2002, Portavella et al 2004). In the experiment conducted by Portavella et al, they damaged the telencephalon of goldfish which caused profound inhibitory avoidance learning response deficit. The results suggested that teleost telencephalon is involved in information storage and further more in learning and memory. Further studies elucidated dorsal telencephalon is involved in learning and memory, especially the dorsal lateral (Dl) and dorsal medial (Dm). For example, the lesion at Dl region of goldfish telencephalon caused deficit in spatial learning (Portavella et al 2002), whereas the lesion at Dm region impaired avoidance conditioning (Portavella et al 2004). More and more studies have confirmed teleost dorsal pallium plays a critical role on learning and memory. In fact, these changes in spatial, emotional response after damaging Dl and Dm division resembled to those of damaging hippocampus and amygdala in amniotes. These results suggested that Dl and Dm division of teleost fish are functionally highly homologous to mammalian hippocampus and amygdala (Braford 1995, Maximino et al 2013). Moreover, the neural mechanism of learning and memory of teleost is similar to that of land vertebrates. . 16 .

(18) 1.4. Synaptic plasticity Synaptic plasticity can be defined as the modification of connection in strength and response between neurons and is considered as a critical neurochemical basis underlying learning and memory process. Synaptic plasticity can be classified according to the period of synaptic efficiency. Short-term synaptic plasticity can last for periods ranging form milliseconds to minutes whereas long-term plasticity last for periods ranging form minutes to several hours, or even weeks (Nam et al 2004, Ng et al 2012).. 1.4.1 Long-term potentiation (LTP) Long-term potentiation (LTP) is the enhancement of synaptic efficiency between two neurons. Large amount of neurotransmitter were released at presynaptic neuron to postsynaptic neuron, triggering a chain of signal transduction. It is a mechanism widely used to discuss learning and. memory. in. electrophysiological. studies. of. amygdala. and. hippocampus in vivo and in vitro. Terje Lømo first observed LTP in the dentate gyrus of rabbit in 1966 by stimulating presynaptic fiber to record the potential shifting of postsynaptic neuron. They discovered that if high frequency stimulation were given to presynaptic fiber, the potential of postsynaptic dentate gyrus would enhance and last for a period of time, which was called long-lasting potentiation (Bliss & Gardner-Medwin 1973, Bliss & Lomo 1973). LTP can be classified into two major groups according to their induction. The most common means of LTP induction . 17 .

(19) is applying series of high-frequency electrical stimulation (HSF) to the excitatory synapses of CA1 region of hippocampus (Wu et al 2001), which. causes. the. activation. of. α-amino-3-hydroxy-5-methyl-4-. isoxazolepropionic acid (AMPA) receptor on postsynaptic neuronal membrane. The activation impels sodium ion entering postsynaptic neuron then neurons undergo depolarization. Later, magnesium ion on N-methyl-D-aspartate (NMDA) receptor left, causing the activation of NMDA receptor. At this time, large amount of calcium ions pump into the cell, making intracellular calcium level rise (Lynch et al 1983, Kakegawa et al 2002, Jin & Hawkins 2003, Nam et al 2004). Intracellular calcium ion can activate protein kinase and phosphatase, enhancing synaptic efficacy. High level of calcium ion level also will activate calcium/calmodulin-dependent protein kinase Ⅱ. (CaMK Ⅱ ) and. protein kinase A (PKA), further influence genetic performance (Collingridge et al 1992) and LTP induction (Kovacs et al 2007). In the end, activation of NMDA receptors leads to synaptic plasticity and learning process. The influx of calcium ion to postsynaptic NMDAR is considered the first step in HFS-induced LTP. In addition, CaMK. Ⅱ. participates in the inhibition of protein phosphatase 2A (PP2A), which prevents the phosphorylation of CaMK Ⅱ. It was considered related to the induction and maintenance of LTP (Huang et al 2001, Kikuchi et al 2003). NMDA receptors are widely distributed in mammalian brains, usually in the areas related in learning and memory, such as hippocampus . 18 .

(20) and amygdala (Marsden 2013). Also previous studies indicated that competitive antagonist of NMDA, D-AP5, was able to inhibit LTP induced by HFS (Harris et al 1984, Morris et al 1986, Morris 2013). Another means of LTP induction is applying forskolin (FSK), which is the activator of adenylyl cyclase. By triggering the trafficking of AMPAR to maintain synaptic efficacy for a long period of time, LTP was induced (Greengard et al 1991, Lu et al 1999, Otmakhov et al 2004). Over the past few years, rodents have been used as a model to conduct experiments related to the formation of LTP. Recently, increasing studies have focused on specific regions of adult zebrafish brain, such as olfactory bulb (Kim et al 2004, Bundschuh et al 2012) and hindbrain. (Hsieh. et. al. 2014,. Yao. et. al. 2014). to. conduct. electrophysiological studies.. 1.4.2 Long-term depression (LTD) Long-term depression (LTD) is the opposite effect to LTP, meaning the reduction in synaptic strength between neurons which is involved in formation of learning and memory (Gaiarsa & Ben-Ari 2006). LTD was first observed in Schaffer collaterals of hippocampus CA3 region and synapse between pyramidal cells of CA1 region. Different from the induction of LTP, LTD was induced by giving low frequency stimulation (LFS, 1-5 Hz for 10-15 minutes) at Schaffer collaterals (Pockett et al 1990). There are similarities between the . 19 .

(21) formation of LTP and LTD, both of which need the activation of NMDA receptors and in influx of calcium ion to postsynaptic neuron. The main difference between the two is the amount of influx of calcium ion. Small amount of calcium ion influx lead to LTD and large amount of calcium influx trigger LTP. In the formation of LTP, calcium ion activates CaMK Ⅱ, later CaMK Ⅱ phosphorylates target protein. As for the formation of LTD, phosphatase that is activated by calcium ion removes phosphate on target molecule. LTD can be classified according to different receptors, including NMDARs and metabotropic glutamate receptors (mGluRs), which induce NMDAR-LTD and mGluR-LTD, respectively. Both NMDARs and mGluRs are the most common receptors that trigger LTD, while NMDAR-LTD is induced by applying LFS (1-5 Hz for 5-20 minutes) (Akhondzadeh & Stone 1996, Debanne & Thompson 1996) and the induction of mGluR-LTD requires the activation of mGluRs by applying groupⅠmGluR agonist, 3, 5-dihydroxyphenylglycine (DHPG) (Brager & Johnston 2007). The induction of mGluR-LTD does not involve the influx of calcium ion (Fitzjohn et al 2001) or participation of serine/threonine protein phosphatase. Rather, LTD formation involves tyrosine protein phosphatase (Moult et al 2006). The activation of NMDARs and mGluRs cause rapid internalization of postsynaptic AMPA receptors, which decrease excitatory transmission and is considered related to the formation of LTD in hippocampus and cerebellum (Silkis 2000, Massey & Bashir 2007). . 20 .

(22) 2. Research aim In order to study the exact role of dorsal telencephalon in zebrafish plays on learning, and compare the similarities and differences of Dl division to contralateral side of Dm division and the neurotransmitter involved in, we used multi-electrode dish 64-channel system (MED64) to assess Dl-Dm pathway of ipsilateral side and contralateral side and synaptic plasticity. Briefly, this study was divided into three stages and each specific aids are summarized as following: 1. Using multi-electrode dish 64-channel system to assess Dl-Dm pathway of ipsilateral side and contralateral side. 2. Using multi-electrode dish 64-channel system to assess synaptic plasticity of Dl-Dm pathway of ipsilateral and contralateral side. 3. Compare the similarities and difference of synaptic transmission of ipsilateral and contralateral Dl-Dm pathway. . 21 .

(23) 3. Materials and methods 3.1. Experimental animals Zebrafish (AB strain) we used in this study were bred in the vivarium (National Taiwan Normal University) with the photoperiod of 14 h light/10 h dark. Fishes were fed once to twice a day and the temperature of water was fixed at 26-28 ℃. Spawns were kept in an 8x8 cm plastic box and breeding methods were consulted from Zebrafish Book (Westerfield, 1995). Experimental procedures were approved by National Taiwan Normal University Animal Care and Utilization Committee (IACUC).. 3.2. Brain slice preparation Adult zebrafish were sacrifice by euthanasia in 0~4 ℃of artificial cerebrospinal fluid (aCSF) solution. aCSF contains 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. Zebrafish brains were rapidly removed in aCSF solution and were immersed in a 4 % low-melting-point agarose (MDBio, Inc., Taiwan). Transverse telencephalic sliced (350 µm) (Ng et al 2012) were obtained using vibratome (MA752, Campden Instruments Ltd., UK) and microscope. Brain slices were incubated in artificially oxygenated (95 % O2/5 % CO2) (Nikinmaa 2002) aCSF solution for at least 1 hour for stabilizing prior to recording.. . 22 .

(24) 3.3. Electrophysiological recording In this study, we used multi-electrode dish 64-channel (MED64) sustem (Alpha MED Sciences, Tokyo, Japan) to record extracellular population spikes. The MED-P515A probe (Alpha MED Sciences, Tokyo, Japan) was used and there are 64 microelectrodes (50 µm x 50 µm) arranged in an 8 x 8 grid with an inter-electrode spacing of 150 µm. In order to increase the cellular adhesion, the surface of MED64 probe was treated with 0.1％ polyethyleneimine (Sigma, St. Louis, MO, USA) dissolved in 25 mM borate buffer (pH 8.4) in room temperature overnight before use. Before electrophysiological recordings, a brain slice was carefully moved into the recording MED64 probe and perfused with the aCSF (28 °C) using a pump (Gilson Minipuls 3, Villiers Le Bel, France). A nylon mesh and a stainless steel wire were used to prevent the shift of brain slice and also increasing the contact with electrodes. One of the 64 microelectrodes was choose as stimulating cathode in Dl division and the rest of the 63 microelectrodes serve as recording cathode. Biphasic rectangular current pulses (0.2 ms duration) were applied every 20 s and the recording cathode was placed at contralateral side of Dm. In this study, the maximum population spike response was defined by giving increasing stimulus intensity until reaching an asymptotic limit. And the current that trigger about 30 to 50 % of maximal responses was used throughout the experiment. Generally, electrophysiological measures of basal synaptic transmission include input-output (I/O) functions and . 23 .

(25) short-term plasticity (paired pulse facilitation, PPF). I/O curves were obtained from 9 incremental stimulation intensities. 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. LTP was induced by applying 5 trains of 1 Hz (2 sec) HFS, the interval between each train was 3 minutes.. 3.4. Drug application Drugs were all prepared from stick solution and diluting in aCSF. Then, the solution was applied to brain slices by suprafusion. 2-amino-5-phosphopentanoate (D-AP5) and 3,5-dihydroxyphenylglycine (DHPG) were both purchased from Abcam, UK and made up in distilled water. 30 µM D-AP5 solution was prepared fresh from 1 mM stock solution and 40 µM DHPG was prepared from 1 mM stock solution as well.. 3.5. Real-time polymerase chain reaction (q-PCR) In order to investigate the distribution of NMDA receptor in left and. right side of telencephalon, telencephalon was rapidly removed from adult zebrafish and separated into left and right hemisphere. Trizol reagent (Invitrogen, Carlsbad, CA) was used to extract mRNA and homogenization. Complimentary DNA (cDNA) was produced from reverse transcription of RNA using reverse transcriptase Ⅲ (Invitrogen). . 24 .

(26) Sample cDNA, primer pair mix, and LightCycler 480 SYBR Green I Master mixture were used to detect PCR products using LightCycler® 480 Roche and LightCycler 480 software release 1.5.0 SP4 to detect fluorescence and finally to detect the distribution of NMDA receptor in left and right hemisphere of telencephalon. EF1α gene was used as internal control.. RNA extraction 10 telencephalons are separated into left and right hemisphere and. put in an eppendorf separately. Add 100 µl Trizol to homogenized tissue and extra 400 µl Trizol was added until the tissue is completely dissolved in solution. Add 100 µl Chloroform, mixed well and left on the ice for 10 mins. Then, centrifuge samples at 13000 rpm for 30 minutes at 4 °C and there were different phases visible within the tube. Transfer the aqueous phase to fresh tube and add equal volume of Isopropanol (aqueous phase : Isopropanol = 1 : 1). Mix thoroughly and store in -20 °C refrigerator overnight.. RNA reverse transcription Centrifuge samples at 13000 rpm for 15 minutes at 4 °C and RNA. pallet can be seen in the bottom. Remove supernatant and add 500 µl EtOH. Flip gently until pallet float. Centrifuge samples at 13000 rpm for 5 minutes at 4 °C and then remove EtOH and allow remaining EtOH to air dry until pallet becomes transparent. Add 20 µl DEPC water and . 25 .

(27) pipetting then quantify each sample using NanoDrop ND-1000(Thermo Scientific). . Mix the following items after finishing the process above. ddH2O. 11-X µl. Random primer (50 ng/λ). 1 µl. 10mM dNTP MIX. 1 µl. Total RNA (4-5 µg). X µl. Place in PCR machine at 65 °C for 5 minutes and place on ice for 1 minute, then add the followings in each tubes. 5X First-strand buffer. 4 µl. 0.1 M DTT. 1 µl. DEPC. 1.5 µl. SuperScript TM III RT (200 units/λ). 0.5 µl. The volume of each tube will be 20µl. Place in PCR machine at 55 °C for 120 minutes, 72°C for 25miutes,and stay at 25 °C. Add 10µl sample and 500µl RDW (dilute to 50X) then store in -20 °C refrigerator. . 26 .

(28) Real-time PCR Mix the following items and put in real-time PCR machine SYBR Green dye. 5 µl. Primer - forward. 0.5 µl. Primer - reverse. 0.5 µl. cDNA. 4 µl. Real-‐time PCR primer sequence 5’ ! 3’ grin1a. Primer sequence. Forward. GCATATAAGCGCCATAAAGACG. Reverse. GTCTGTGGGTGGGTATTGCT. grin1b. Primer sequence. Forward. TATCAACAGGAGCGAGCGTC. Reverse. GTGTCCGTTGATGAGGCAGA. EF1α. Primer sequence. Forward. CCTCTTTCTGTTACCTGGCAAA. Reverse. CTTTTCCTTTCCATGATTGA. . 27 .

(29) Real-‐time PCR temperature 95 ℃. 20 s. 95 ℃. 3s. 60 ℃. 30 s. 95 ℃. 5s. 65 ℃. 1 min. 97 ℃. x. 40 ℃. 30 s. 1 cycle. 45 cycle. 1 cycle. 1 cycle. 3.6. Statistical analysis All experiment values were expressed as mean ± S.E.M. D-AP5 experiment values were tested using one-way ANOVA (Dunnett’s test). The rest of data were tested using paired t-test. In all cases, p<0.05 was considered to be significant. Statistical analysis was performed using PRISM version 6.0 . 28 .

(30) 4. Results 4.1. Excitatory postsynaptic potentials in Dm division of the telencephalon recorded by multi-electrode arrays (MED64) For electrophysiological recordings, we placed a single 350 µm thick brain slice on the recording MED probe (Fig. 1A). A bipolar stimulation was given to Dl division, located at the border between posterior (Dp) zones and sulcus ypsiloniformis (Y) (Fig. 1B) and a negative peak was evoked in Dm division in both ipsilateral and contralateral side. We characterized neuronal properties by means of input-output response, such as input-output (IO) curve and pair-pulse facilitation (PPF). We recorded a population spike in the Dm division of contralateral side by giving stimulation in the Dl division. As shown in Fig. 1C, the amplitude of population spike in the Dm division of contralateral side gradually increased from threshold value with the rising amplitude of stimulation until smoothly saturated form. In Fig. 1D, Dm division of contralateral side exhibited paired pulse facilitation, which paired pulse stimulation produced facilitation in response to the second stimulus.. . 29 .

(31) 4.2. Dl-evoked long-term potentiation (LTP) in Dm division of contralateral side High frequency stimulation (HFS)-induced LTP. Experiment 1 1. Research aim: Previous study has described the dorsal telencephalic synaptic plasticity (Ng et al 2012), especially the Dl-Dm pathway in the zebrafish, including LTP and LTD two forms. The formation of LTP and LTD are related to N-methyl-D-aspartate receptor and metabotropic glutamate receptors (mGluRs) respectively. In this study, we aim to investigate whether stimulation in Dl division will evoke synaptic plasticity in the Dm of contralateral side in dorsal telencephalon. 2. Experimental procedure: As described in material and methods, we used AB strain zebrafish aged 3 to 5 months, and their brains were removed and sliced into 350 µm thick telencephalic slices for in vitro electrophysiological recording. First, stimulation (0.2 ms) was given every 20 seconds for 10 minutes as baseline recording and 5 times of high frequency stimulation (100 Hz) was applied. The interval of each trial is 3 minutes. The recording lasted for another 1 hour to observe the synaptic plasticity in the Dl-Dm of contralateral side pathway.. . 30 .

(32) 3. Results: The results were shown in Fig. 2. Following a HFS given in Dl division, LTP can be induced in Dm division of ipsilateral and contralateral side. The amplitude of population spike of both sides of LTP was 1.5 times lager than the amplitude of baseline and can last at least 1 hour.. The amplitude of population spike of the contralateral Dm at 1. hour after HFS was 204 ± 11.8 (n=16, p<0.0001); the amplitude of population spike of the ipsilateral Dm at 1 hour after HFS was 208 ± 41.9 (n=16, p<0.05). From the result, we can infer that applying HFS at one side can induce LTP in Dm division of ipsilateral and contralateral side.. . 31 .

(33) Experiment 2 1. Research aim: Previous studies inferred that the formation of LTP depends on the activation of NMDA receptor. Thus, this experiment will investigate whether Dl-Dm of contralateral side pathway depends on NMDA receptors as well. We applied NMDA receptor antagonist, D-AP5, to observe the influence on LTP induction. 2. Experimental procedure: As described in material and methods, we used AB strain zebrafish aged 3 to 5 months, and their brains were removed and sliced into 350 µm thick telencephalic slices for in vitro electrophysiological recording. First, stimulation (0.2 ms) was given every 20 seconds for 10 minutes as baseline recording and 30 µM of D-AP5 was applied for 30 minutes. The first HFS (arrow) was applied 15 minutes after application of D-AP5 and the second HFS was applied 30 minutes after D-AP5 had been washed out. The recording lasted for another 1 hour to observe the synaptic plasticity in the Dl-Dm of contralateral side pathway. 3. Results: The results were shown in Fig. 3. The amplitude of population spike of the contralateral Dm at 15 minutes after application of D-AP5 was 87 ± 5.1 (n=9, p>0.05); the amplitude of population spike of the ipsilateral Dm at 15 minutes after application of D-AP5 was 104 ± 10.3 . 32 .

(34) (n=9, p>0.05), suggesting that application of 30 µM of D-AP5 alone did not produce any significant changes. Under the effects of D-AP5, Dm divisions of ipsilateral and contralateral sides were unable to induce LTP. The amplitude of population spike of the contralateral Dm at 30 minutes after the first HFS was 100 ± 6.5 (p>0.05); the amplitude of population spike of the ipsilateral Dm at 30 minutes after the first HFS was 100 ± 8.4 (p>0.05). When the second HFS was delivered after the washout of D-AP5, LTP can be induced successfully. The amplitude of population spike of the contralateral Dm at 10 minutes after the second HFS was 224 ± 30.9 (p<0.05); the amplitude of population spike of the ipsilateral Dm at 10 minutes after the second HFS was 240 ± 34.2 (p<0.05). 60 minutes after the second HFS, the amplitude of population spike of the contralateral Dm was 194 ± 29.5 (p<0.05); the amplitude of population spike of the ipsilateral Dm was 168.2 ± 11.5 (p<0.05). The results suggested that HFS-induced LTP needed the activation of NMDA receptors in the contralateral Dm.. . 33 .

(35) Experiment 3 1. Research aim: From our experiment we observed that HFS given from Dl division of left or right hemisphere could induce LTP in the Dl-Dm pathway of both ipsilateral and contralateral side. This experiment will further analyze the differences between HFS given from Dl of left or right hemisphere and differences of synaptic plasticity between Dl-Dm pathway of ipsilateral and contralateral side. 2. Results: The results were shown in Fig. 4 and Fig. 5. As shown in Fig. 4, the amplitude of population spike of Dm of ipsilateral and contralateral side following stimulation from Dl of the left hemisphere was stronger than the stimulation given from the right hemisphere. In Fig. 4A, at the early phase of LTP (5-15 mins after HFS), the amplitude of population spike of contralateral Dm, following HFS given from the Dl of left hemisphere showed the trend of stronger than stimulation given from the right hemisphere, but wasn’t statistically significant (stimulation from the left=8, stimulation from the right, n=8 (p=0.24)); in Fig. 4B, the early phase (5-15 mins after HFS), the middle phase (25-35 mins after HFS) and the late phase (45-55 mins after HFS) of LTP, the amplitude of population spike of ipsilateral Dm, following HFS given from the Dl of left hemisphere was stronger than stimulation given from the right hemisphere. The difference of early phase LTP was statistically . 34 .

(36) significant. (Stimulation from the left=8, stimulation from the right, n=8 (p<0.24)) but the middle phase (p=0.07) and late phase (p=0.09) weren’t. In Fig. 5, we observed that the stimulation given from Dl of left or right hemisphere, the amplitude of population spike of contralateral Dm was stronger than the one of the ipsilateral side. In Fig. 5A, following stimulation given from Dl of left hemisphere has no significant differences with ipsilateral side (n=8, p=0.3); in Fig. 5B, following stimulation given from Dl of right hemisphere, the early phase (p<0.01), middle phase (p<0.01), late phase (p<0.01) and 60 min after HFS, the amplitude of population spike of contralateral Dm were stronger than the one of ipsilateral side. The results inferred that the mechanism underlying synaptic plasticity of left and right hemisphere of zebrafish telencephalon was different. . 35 .

(37) 4.3. Dl-evoked long-term depression (LTD) in Dm division of contralateral side 4.3.1. Low frequency stimulation (LFS)-induced LTD. 1. Research aim: The experiment will investigate another form of synaptic plasticity, long-term depression. LFS-induced LTD is related to the amount of calcium release. In this study, we aim to investigate whether stimulation in Dl division will evoke synaptic plasticity in LTD form in the Dm division of contralateral side. 2. Experimental procedure: As described in material and methods, we used AB strain zebrafish aged 3 to 5 months, and their brains were removed and sliced into 350 µm thick telencephalic slices for in vitro electrophysiological recording. First, stimulation (0.2 ms) was given every 20 seconds for 10 minutes as baseline recording and applied low frequency stimulation (1 Hz) for 20 minutes. The recording lasted for another 1 hour to observe the synaptic plasticity in the Dl-Dm of contralateral side pathway. 3. Results: The results were shown in Fig. 6. Following a LFS given in Dl division, LTD can be induced in Dm division of ipsilateral and contralateral side. The population spike amplitude of LTD of both sides . 36 .

(38) was 80 % smaller than the amplitude of baseline and can last at least 1 hour.. The population spike amplitude of contralateral Dm at 1 hour. after LFS was 77 ± 2.9 (n=11, p=0.09); the population spike amplitude of ipsilateral Dm at 1 hour after LFS was 75 ± 5.3 (n=11,. p=0.15).. From the result, we can infer that applying LFS at one side can induce synaptic plasticity in LTD form in Dm division of both ipsilateral and contralateral side.. . 37 .

(39) 4.3.2. DHPG-induced long-term depression. 1. Research aim: Previous study showed that the application of mGluR agonist, DHPG, could induce LTD in Dl-Dm pathway of zebrafish telencephalon. In this study we aim to investigate whether DHPG could induce synaptic plasticity in LTD form in Dm division of contralateral side. 2. Experimental procedure: As described in material and methods, we used AB strain zebrafish aged 3 to 5 months, and their brains were removed and sliced into 350 µm thick telencephalic slices for in vitro electrophysiological recording. First, stimulation (0.2 ms) was given every 20 seconds for 10 minutes as baseline recording and 40 µM of DHPG was applied for 10 minutes. The recording lasted for 1 hour to observe the synaptic plasticity in the Dl-Dm of contralateral side pathway. 3. Results: The results were shown in Fig. 7. Application of DHPG caused LTD in Dm division of ipsilateral and contralateral side. Both sides of population spike amplitude were 70 % smaller than the one of baseline and can last for at least 1 hour. In Fig. 7C, the comparison on PS amplitude of DHPG-induced LTD in contralateral and ipsilateral Dm showed significant difference between the two in the early phase of LTD . 38 .

(40) (25-35 min). The PS amplitude of contralateral side was smaller than that of ipsilateral side in 25-35 min (n=9, p<0.05). The population spike amplitude of contralateral Dm at 1 hour after application of DHPG was 65 ± 8.1 (n=9, p<0.01); the population spike amplitude of ipsilateral Dm at 1 hour after application of DHPG was 68 ± 2.4 (n=9, p<0.01). The results suggested that application of DHPG induced synaptic plasticity in LTD form in Dm division of both ipsilateral and contralateral side.. . 39 .

(41) 4.4. Characteristics of field potentials of Dl-evoked field potentials in Dm division of contralateral side Excitatory postsynaptic potentials in Dm division of contralateral side of the telencephalon 1. Research aim: In this experiment we discussed the characteristic of neuron transduction in Dl-Dm of contralateral side pathway by relationship of stimulation and response, such as input-output (IO) curve and paired pulse facilitation (PPF). 2. Experimental procedure: As described in material and methods, we used AB strain zebrafish aged 3 to 5 months, and their brains were removed and sliced into 350 µm thick telencephalic slices for in vitro electrophysiological recording. IO curve started the stimulation at -5 μV and added -5 μV after 3 repetition until smooth saturation. Stimulation of PPF was giving 2 continuous stimulations, and the intervals of 2 stimulations were 20 ms, 50 ms, 100 ms, 150 ms and 200 ms. 3. Results: The results were shown from Fig. 8 to Fig. 13. In IO curve of Fig. 8, the amplitude of population spike gradually increased from threshold following the increasing stimulation intensity until smoothly saturated in Dm of both ipsilateral and contralateral side. In Fig. 9A, the amplitude of . 40 .

(42) population spike of contralateral side by stimulation from the right side showed the trend to be stronger than stimulation from the left side while the amplitude of population spike of ipsilateral side had no significant differences in Fig. 9B. In Fig. 10A and Fig. 10B, the comparison of contralateral side and ipsilateral side following stimulation from both side showed no significant differences. Fig.11 showed PPF evoked in Dm of contralateral and ipsilateral side. In Fig. 12A, the paired pulse ratio of PPF of contralateral side by stimulation from the right side showed no significant differences with stimulation from the left side while in Fig. 12B, the paired pulse ratio of PPF of ipsilateral side by stimulation from the right side was larger than that by stimulation from the left side at the interval of 150 ms. In Fig. 13A, pair pulse ratio of contralateral side was significantly larger than that of ipsilateral side following stimulation from the left side at the interval of 20 ms. In Fig. 13B, pair pulse ratio of contralateral side and ipsilateral side following stimulation from the right showed no significant differences. The mechanism for pair pulse facilitation is due to the presynaptic increasing amount of calcium ion, causing more neurotransmitter to release. PPF produced a facilitation of field potential in response to the second stimulation. Paired pulse ratio refers to the second field potential area divide the first field potential area. (Contralateral hemisphere: 20 ms: 239.6 ± 9.2 (p<0.01); 50 ms: 201.5 ± 8.9 (p<0.01); 100 ms: 164.1 ± 9.8 (p<0.01); 150 ms: 140.3 ± 10 (p<0.01); 200 ms: 134.1 ± 10.1 (p<0.01); (ipsilateral hemisphere: 20 ms, 213.7 ± . 41 .

(43) 11.6 (p<0.01); 50 ms, 186.9 ± 10.9 (p<0.01); 100 ms, 157.2 ± 10 (p<0.01); 150 ms, 141.7 ± 11.1 (p<0.01); 200 ms, 125.2 ± 7.9 (p<0.01).. . 42 .

(44) 4.5. NMDA receptor distribution of left and right hemisphere of telencephalon 1. Research aim: In this experiment we discussed the distribution of NMDA receptor subtypes NMDAR1a and NMDAR1b at left and right hemisphere of telencephalon using real-time PCR. 2. Experimental procedure: As described in material and methods, we used AB strain zebrafish aged 3 to 5 months, 10 telencephalons are separated into left and right hemisphere. RNA were extracted and reversely transcript to cDNA. Primers for NMDAR1a and NMDAR1b were designed and used in real-time PCR process. 3. Results: The results were shown in Fig. 14. In NMDAR1a, the mRNA expression of left hemisphere showed the tendency of higher expression than right hemisphere (left hemisphere n=4, p=0.13), whereas NMDAR1b, no difference or tendency was observed between left and right hemisphere (right hemisphere n=4, p=0.39). Right hemisphere of mRNA expression was used as control.. . 43 .

(45) 5. Discussion Cerebral lateralization has been a widely observed phenomenon from fishes (Watkins et al 2004) to mammals (Hosaka et al 2015) though it had been thought to be a unique trait to human at first. The most perceptible traits in human cerebral lateralization including different anatomical brain structure, dominant hemisphere for distinct functions such as language and facial recognition (Keller et al 2011, Okazaki et al 2014), and preferential use of hands (Riolo-Quinn 1991, Moura 2015). Increasing studies indicated the functional lateralization by lesion on unilateral hemisphere, which caused impairment on different aspects including learning and memory (Floel et al 2004, Glikmann-Johnston et al 2008). Given the previous studies are exclusively focused on human patients and mammalian models, with the advantage of easy maintain and molecular manipulation, it is imperative to study the possible cerebral lateralization phenomena in zebrafish models. In the present study, we used electrophysiological approaches and zebrafish model to characterize the synaptic plasticity of contralateral dorsal lateral and dorsal medial pathway within dorsal telencephalon. It is a nice extension of our previous results showed that after given electrical stimulation in the Dl division can evoked a field potential in the ipsilateral Dm division. In addition, both LTP and LTD were induced by applying HFS and LFS, respectively. Our previous results also demonstrated that a glutamatergic neurotransmission is involved in both . 44 .

(46) LTP and LTD formation. Co-administration of NMDA receptor agonist AP5 blocked the formation of HFS induced LTP in Dm division. Either LFS or suprafusion of metabotropic glutamate receptor agonist DHPG induced LTD in the Dm division (Ng et al 2012). In the present study, we expand our research interest on the bilateral neuroplasticity in the Dm region. By giving HFS in either left or right Dl, we induced stable, robust LTP in both Dm division of ipsilateral and contralateral side following stimulation from either left or right Dl. Similar results of LTP induction in contralateral hippocampus of other species were observed including mouse (El-Gaby et al 2015) and guinea pigs (Chirwa et al 2001). Which implies a highly evolutional conservation on the neural mechanism of neuroplasticity is existed among different species. Many of the studies regarding neuronal transduction between two hemispheres use in vivo experiments due to the limitation of brain slice preparation (Shipton et al 2014, El-Gaby et al 2015). The complex structure and neuronal circuit of mammalian brain might be more vulnerable during the process of brain slice preparation due to larger size of brain. Several studies indicated the LTP induction is NMDA-dependent in CA1-CA3 connection of hippocampus (Kanterewicz et al 2000, Otmakhov et al 2004) and zebrafish telencephalon (Nam et al 2004, Ng et al 2012), which is consistent with our data that application of NMDA . 45 .

(47) receptor antagonist, D-AP5 blocked the induction of LTP, while upon washout of D-AP5, LTP could be restored. The similar results are also discovered in LTP induction from Dl division to contralateral Dm division of the telencephalon in zebrafish. Which suggest zebrafish can be used as an alternative model for studying the NMDA dependent neuroplasticity. It had been proven that activation of NMDA receptors triggered long-term potentiation in hippocampal slices of both ipsilateral and contralateral side and is related to the cellular mechanism responsible for synaptic plasticity and resulted in the formation of learning and memory (Beck et al 2000, Knafo et al 2012). As reported in previous studies, by applying specific antagonist of NMDA receptors has demonstrated to cause impairment in spatial learning (Morris 1989) and memory retention (Kim et al 1991) in rodents. In teleost fish, NMDA receptor plays an important role in avoidance learning (Xu et al 2003) and spatial learning (Gomez et al 2006) as well. Interestingly, several studies indicated the allocation of NMDA receptor subunits is asymmetrical in hippocampal CA1-CA3 circuitry, which may produce unequal numbers of NMDA receptor and therefore resulting in distinct ability to express synaptic plasticity (Kawakami et al 2003, Shinohara & Hirase 2009). In present studies, we divided the position of stimulus into left and right hemisphere and discovered that PS amplitude of Dm division in both ipsilateral and contralateral side exhibited stimulus from the left caused greater PS . 46 .

(48) amplitude than that from the right in early stage LTP. While applying a stimulus from the right, PS amplitude of Dm of contralateral side will be larger than Dm of ipsilateral side. Both results indicated that telencephalon of the left hemisphere is dominant than that of right hemisphere in the synaptic plasticity. We speculate the similar asymmetric distribution of NMDA receptors may also occur in the Dl-Dm circuitry of zebrafish telencephalon and therefore caused different ability to express synaptic plasticity in left and right hemisphere of telencephalon. Therefore, we conducted real-time PCR to verify the distribution of NMDAR subtype including NMDAR1a and NMDAR1b. Both subtypes were reported expressed mainly in brains and nervous system (Cox et al 2005, Tzeng et al 2007). Our results suggested that the NMDAR1a mRNA expression of left hemisphere showed the tendency of higher expression than right hemisphere, while NMDAR1b expressed no difference or tendency. The results were undetermined possibly due to insufficient sample number or the dilution of exact NMDA mRNA caused by numerous telencephalons mixed as one sample. Further experiments such as immunohistochemistry and comparison of NMDA-dependent LTP on D-AP5 threshold dose are worth for determining this possibility. Besides bilateral LTP formation, we demonstrated that application of mGluR agonist, DHPG could also triggered a stable, robust LTD in both Dm division of ipsilateral and contralateral side, which was . 47 .

(49) consistent with previous results of DHPG-induced LTD in unilateral hippocampal CA1 region (Palmer et al 1997, Fitzjohn et al 1999, Schnabel et al 1999). We also discovered that the PS amplitude of DHPG-induced LTD of contralateral telencephalon was smaller than that of ipsilateral telencephalon in early phase. However, Studies related to DHPG-induced LTD are scarce and the mechanism underlying LTD asymmetry remains unclear. Future studies related to application of mGluR antagonist or separation of stimulus into different hemispheres would help to clarify the mechanism of LTD induction in different hemispheres. It is well known that the mGluR regulates activity of neuronal ion channels, such as AMPA receptor. A previous study indicated that DHPG-induced LTD was related to the internalization of AMPA receptors, causing the removal of AMPA receptors from the membrane (Xiao et al 2001). Hence, from our results we speculated the molecular mechanism that regulates LTD formation is similar between adult zebrafish and mammals. Further experiments such as comparison of the AMPA receptor expression among the bilateral Dm division will be helpful for testing our hypothesis. Another interesting result from our results is the difference on the latency of initial positive deflection between Dm field potential of contralateral and ipsilateral side, the latency of initial positive deflection of contralateral side lasted longer than that of ipsilateral side, which might be caused by the different distance of stimuli through biological . 48 .

(50) tissue towards recording cathode between contralateral and ipsilateral side. According to our lab’s previous studies, the field potential is composed of non-synaptic (P1) and synaptic (N2) components according to the variance of potential amplitude in intensity-response curve (IO-curve), paired pulse facilitation and application of tetrodotoxin (TTX), a strong inhibitor of voltage gated sodium channels (Ng et al 2012). P1 remained the same in paired pulse facilitation and variant in IO-curve. These results correspond to the observation that a positive deflection fiber volley (FV) waveform was evoked in CA3 division following a stimulation in hippocampal mossy fibers (Henze et al 1997) and so P1 component were identified as fiber volley. In present studies, the field potential recorded in Dm of contralateral and ipsilateral side both exhibited the same phenomenon, which the amplitude of P1 of both sides increased following the increase stimulus intensity in IO-curve while the amplitude of P1 of both sides maintained the same in paired pulse stimulation, suggesting the same results with Ng et al. that P1 component is non-synaptic and N2 is synaptic. An elaboration of this point can be studied by applying Ca2+ and Mg2+ solution to assess ionic channel underlying field potential transduction. A recent study indicated that bilateral hippocampal structure is functionally unequal. Left hippocampus plays a dominant role in spatial short-term and long-term memory, whereas asymmetric synaptic plasticity was discovered in mouse hippocampus as well (Shipton et al . 49 .

(51) 2014). Also, from our lab’s previous studies, unilateral side of telencephalic ablation in zebrafish impaired spatial short-term memory and moreover indicating left side telencephalon is dominant in learning and identifying new objects while right side telencephalon is dominant in long-term memory acquisition, retention and retrieval (Wu 2008). The results provided an evidence of functional lateralization and were correspondent with those of our electrophysiological studies, which implies left hemisphere telencephalon is dominant in learning than left hemisphere. With the progressive medical imaging equipment, increasing numbers of human anatomical and functional evidences involving lesion on unilateral hippocampi caused impairment on spatial memory have shown functional asymmetry between the two hemispheres (Abrahams et al 1997, Glikmann-Johnston et al 2008, Duecker et al 2013). Evidences from cellular mechanism to behavioral aspect are implying the notion of functional lateralization of the hippocampus. In conclusion, our results demonstrated that stimulus in Dl division can evoked extracellular potential in Dm division of both ipsilateral and contralateral side. Dm division of bilateral hemispheres exhibits paired pulse facilitation, long-term potentiation and long-term depression. Most importantly, we discovered the different ability to express long-term potentiation between the two hemispheres of zebrafish telencephalon. . 50 .

(52) Our findings present new electrophysiological evidence in cerebral lateralization and facilitate the use of zebrafish as a model for electrophysiological researches.. . 51 .

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