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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 管永恕(Yung-Shu Kuan) | |
| dc.contributor.author | CHON-LONG MUI | en |
| dc.contributor.author | 梅駿朗 | zh_TW |
| dc.date.accessioned | 2021-07-11T14:58:56Z | - |
| dc.date.available | 2026-02-03 | |
| dc.date.copyright | 2021-03-02 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-02-01 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78473 | - |
| dc.description.abstract | 脊椎動物的韁核（habenula nuclei）是位於間腦，毗鄰松果體（pineal gland）左側和右側。韁核在生理上以及心理上皆扮演很重要的角色，如：調節疼痛﹑獎勵系統﹑焦慮和睡眠等行為。在以往的研究中，發現對小鼠或斑馬魚的韁核進行破壞，會出現睡眠障礙並引起晝夜節律紊亂，嚴重會導致抑鬱症或焦慮症。然而，韁核受損所引起之行為異常在分子層面上仍尚不清楚。 有研究已經證明成年和10天大幼魚的韁核神經元在分化程度上高度相似。因此本研究利用雙側電消融對10天大幼魚的韁核造成損傷然後研究韁核受損後於幼年斑馬魚的腦中之分子變化。 在先前實驗室研究中，在4-6天接受韁核雷射消融的成魚中觀察到睡眠障礙。此外，在先前實驗室研究中亦發現clocka 和chat在消融的成魚全腦中也出現變化。然而，clocka和chat在韁核消融的少年斑馬魚尚未被研究。因此，本研究使用原位雜交(In Situ Hybridization)來研究 clocka 和chat在韁核消融的少年斑馬魚的全腦表現。結果發現chat在內側縱筋膜（mlf）和外側核瓣（nlv）沒有明顯變化。此外clocka在少年消融斑馬魚中腹側端腦和視頂蓋的腦室周圍灰質中出現上調。這些分子變化可能是韁核損傷引起的脊椎動物睡眠障礙的原因。 為了進一步鑑定由韁核受損於幼年斑馬魚的基因變化，我的實驗是針對韁核受損和未經處理的幼魚的全腦進行了核糖核酸測序（RNA-sequencing）。測序結果顯示有6個參與晝夜節律途徑的基因產生了變化。為了驗證核糖核酸測序的結果，本研究利用反轉錄即時聚合酶連鎖（qRT-PCR）反應，發現per1a﹑per1b和 per3出現明顯的上升。但在原位雜交(In Situ Hybridization)染色結果比較中發現,韁核受損和和未經處理的幼魚的全腦在這三個基因的表現上並沒有明顯的差異。 除了探究分子變化外，本研究還使用這種雙側韁核電消融斑馬魚模型進行再生藥物之初探，初步測試了孕烯醇酮（Pregnenolone;P5），當歸（Radix Angelicae Sinensis）以及乳香（Frankincense）在韁核受損後再生的功效和行為的影響。 在新魚缸實驗(NTD)中經1微摩爾濃度（uM）的孕烯醇酮，每毫升1000毫克(1000mg/ml)的當歸處理後沒有明顯影響斑馬魚的行為，而每毫升150毫克(150mg/ml)的乳香（Frankincense）處理後使正常斑馬魚花更多時間在魚缸底部，表明乳香處理後使斑馬魚感到更焦慮。而經1微摩爾濃度（uM）的孕烯醇酮，每毫升1000毫克(1000mg/ml)的當歸及，每毫150毫克(150mg/ml)的乳香處理後在韁核再生方面沒有作用。 | zh_TW |
| dc.description.abstract | The vertebrate habenular nuclei are located in the dorsal diencephalon, adjacent to the left and right sides of the pineal gland. It plays an important role in regulating pain, reward, anxiety, and sleep. Many studies have indicated that destruction of the rat and zebrafish habenula led to disconcerted circadian rhythms and resulted in sleep disturbance, as well as depression and anxiety. However, the molecular alterations of habenula ablation zebrafish remained less understanding. Habenula neuronal differentiation is found to be highly similar between adults and larvae at 10 days post fertilization (10 dpf). Therefore, electrolytic ablation is used to create bilateral habenula damages on 10 dpf larvae and the post-ablation molecular alterations of 42 dpf juvenile zebrafish are investigated. Our previous finding indicated that sleep disturbance is exhibited in 3 to 6 month-old adult zebrafish whose habenula are ablated at 4-6 dpf. Moreover, the clock circadian regulator a (clocka) and choline acetyltransferase (chat) are altered in habenula-ablated adult zebrafish. However, the alterations of clocka and chat have not been reported in habenula-ablated juvenile zebrafish. Therefore, ISH is used to investigate the expressions of clocka and chat in the whole brain of habenula-ablated juvenile zebrafish. The results from whole brain sections showed that no significant chat expression difference in medial longitudinal fasciculus (MLF) and nucleus lateralis valvulae (NLV). And the results from whole brain sections showed that clocka expression was upregulated in ventral telencephalic and periventricular grey zone (PGZ) of the tectum opticum (TeO). These alterations may be associated with the sleep disturbance observed in mammal with habenular impairment. Whole brain RNA sequencing is used to further investigate the alteration of gene expression in habenula-ablated juvenile zebrafish. The results demonstrated that the 6 circadian rhythm genes are altered after electrolytic ablation in juvenile stage. The expressions of per1a, per1b and per3 are upregulated in habenula-ablated juvenile zebrafish compared with the control by qRT-PCR. However, In situ hybridization (ISH) results show that no significant difference in per1a, per1b and per3 expression in the whole brain. In addition , the effects of Pregnenolone (P5), RAS (當歸) and Frankincense (乳香) on habenula regeneration and behavior alteration are also investigated in this study. In the NTDT, there are no notably behavioral difference in zebrafish after P5 and RAS treatments. Surprisingly, the control group spent less time in the tank’s upper ½ portion after the 150μg/ml Frankincense treatment. This suggested that 150ug/ml Frankincesnse treatment may increase anxiety behavior. On the other hand, ablated zebrafish treated with 1uM P5,150 μg /ml Frankincense and 1000 μg /ml RAS for 2 hours once on every other day until 39dpf showed no effect on the regeneration of habenula. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T14:58:56Z (GMT). No. of bitstreams: 1 U0001-2501202117223300.pdf: 2275055 bytes, checksum: 571b0df668ed8c1e10fba20204e2dec5 (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | List of Abbreviations III 中文摘要 VI Abstract VIII Table of content XI Chapter 1 Introduction 1 1.1 Habenula-Interpeduncular circuit 1 1.2 Habenula in emotion disorder 1 1.3 Habenula in sleep and circadian rhythm 2 1.4 Molecular alteration in habenula lesion adult zebrafish 3 1.5 Zebrafish-based drug screening and drug 4 1.5.1 Pregnenolone(P5) 5 1.5.2 Radix Angelicae Sinensis (當歸) 6 1.5.3 Frankincense(乳香) 7 1.6 Aim of this study 8 Chapter 2 Material and Methods 9 2.1 Zebrafish 9 2.2 Bilateral Electrolytic Ablation of Habenula 9 2.3 Confocal Microscopy Imaging Analysis 9 2.4 Fixation and Pigment removal of Zebrafish Larvae 10 2.5 Dissection and Fixation of Zebrafish Brain 10 2.6 RNA Extraction and RNA Sequence Analysis 11 2.7 Quantitative reverse transcription PCR (qRT-PCR) 11 2.8 Probe Synthesis 12 2.9 In situ hybridization 12 2.9.1. zebrafish larvae 12 2.9.2. Juvenile zebrafish brains tissue 14 2.10 Pregnenolone(P5) treatment 15 2.11 Radix Angelicae Sinensis (當歸)treatment 15 2.12 Frankincense(乳香)treatment 16 2.13 Novel tank diving test (NTD) 17 Chapter 3 Results 18 3.1 Juvenile zebrafish show low regeneration rate after 10dpf bilateral electrolytic ablation 18 3.2 Bilateral electronic ablation shows no significant difference in choline acetyltransferase (chat) expression in rhombencephalon 19 3.3 The expression of clock circadian regulator a (clocka) is upregulated in ventral telencephalon (VT) and the periventricular grey zone (PGZ) of the optic tectum (TeO) 19 3.4 Circadian rhythm genes were altered after bilateral electrolytic ablation 20 3.5 The expression of per1a, per1b and per3 show no significant difference in the whole brain section 21 3.6 Control zebrafish treated with Frankincense (乳香) spent more time in the bottom 22 3.7 Drug treatment of Pregnenolone(P5), radix angelicae sinensis (當歸) and Frankincense(乳香) cannot enhance the regeneration of habenula 23 Chapter 4 Conclusions and Discussion 25 References 31 Figures 37 Supplementary Figures 56 | |
| dc.language.iso | en | |
| dc.subject | 行為測試 | zh_TW |
| dc.subject | 韁核 | zh_TW |
| dc.subject | 雙側電消融 | zh_TW |
| dc.subject | 晝夜節律 | zh_TW |
| dc.subject | 斑馬魚 | zh_TW |
| dc.subject | 韁核再生 | zh_TW |
| dc.subject | bilateral electrolytic habenula | en |
| dc.subject | habenula | en |
| dc.subject | behavior test | en |
| dc.subject | habenula regeneration | en |
| dc.subject | zebrafish | en |
| dc.subject | circadian rhythm | en |
| dc.title | 韁核損傷對睡眠干擾之機制與再生藥物之初探 | zh_TW |
| dc.title | Molecular Mechanism Underlying Habenula Damage-Induced Sleep Disturbance and Preliminary Exploration of Drugs on Habenula Regeneration | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 109-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 黃聲蘋(Sheng-Ping Hwang),胡清華(Chin-Hwa Hu),蕭崇德(Chung-Der Hsiao) | |
| dc.subject.keyword | 韁核,雙側電消融,晝夜節律,斑馬魚,韁核再生,行為測試, | zh_TW |
| dc.subject.keyword | habenula,bilateral electrolytic habenula,circadian rhythm,zebrafish,habenula regeneration,behavior test, | en |
| dc.relation.page | 64 | |
| dc.identifier.doi | 10.6342/NTU202100162 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2021-02-02 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| dc.date.embargo-lift | 2026-02-03 | - |
| 顯示於系所單位： | 生化科學研究所 | |
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