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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王致恬(Chih-Tien Wang) | |
dc.contributor.author | Yung-Chi Chang | en |
dc.contributor.author | 張詠淇 | zh_TW |
dc.date.accessioned | 2021-06-16T13:29:58Z | - |
dc.date.available | 2018-07-26 | |
dc.date.copyright | 2013-07-26 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-07-22 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/62140 | - |
dc.description.abstract | 下視丘-腦下垂體系統由magnocellular neurons (MCNs)所組成,製造神經傳導物質(催產素和血管加壓素),分別沿著神經元的樹突釋放到腦脊髓液中或是沿著軸突釋放都周邊血液系統中。在下視丘-腦下垂體系統中有兩種囊泡包裹神經傳導物質,分別是large-dense core vesicles (LDCVs)和microvesicles (MVs)。先前研究指出一種弱鈣離子感應蛋白-Synaptotagmin IV (Syt IV)表現在LDCVs和MVs中,並且參與鈣離子調控性胞吐作用。然而在下視丘-腦下垂體系統中,MVs不論在大小、型態和分子組成上與其他神經組織中的synaptic vesicles (SVs)相類似,但MVs其包裹的神經傳導物質和在下視丘-腦下垂體系統中的功能尚未了解。因此,我們假設在成年雄鼠的下視丘-腦下垂體系統中,一種SV/MV的蛋白-Synapsin Ia (Syn Ia)可能會影響神經胜肽從LDCVs中釋放。首先,我們利用免疫螢光染色,可以觀察到Syn Ia在視上核和腦下腺後葉的表現不會和包覆在LDCVs中的催產素和血管加壓素重疊,證明Syn Ia 是在MVs中表現。接著,我們利用活體電穿孔方式,將不同種的DNA轉染到視上核中。轉染的DNA種類包含Syt I、 Syt IV、wild-type Syn Ia和仿效無法被磷酸化的Syn Ia (Syn Ia-S62A). 我們利用酵素連結免疫吸附試驗,來偵測催產素和血管加壓素在腦脊髓液和周邊血液中的濃度,因為轉染不同種DNA而造成的影響。研究結果顯示,Syn Ia 促進催產素在腦脊髓液和周邊血液中的含量增加,並且這個情形會因為Syn Ia-S62A而反轉。當共同轉染Syt IV和Syn Ia時,仍然會增加催產素在腦脊髓液和周邊血液中的含量,並且一樣會被共同轉染Syt IV 和Syn Ia-S62A反轉。因此,證明Syn Ia是藉由第62個胺基酸的位置磷酸化與否來調控催產素在腦脊髓液和周邊血液中的釋放。另一方面,血管加壓素釋放到腦脊髓液和周邊血液中,也是藉由Syn Ia第62個胺基酸的位置磷酸化來調控。此外,鈣離子感應蛋白-Syt I會增加血管加壓素在周邊血液的釋放量,並且會被Syt IV所反轉。綜合上述,可以得知在下視丘-腦下垂體系統中,Syn Ia調控催產素和血管加壓素的釋放是藉由第62個胺基酸的位置磷酸化與否,並且仿效無法被磷酸化的Syn Ia會抑制MV/SV的傳遞,來調控LDCVs的釋放。 | zh_TW |
dc.description.abstract | The magnocellular neurons (MCNs) of hypothalamic neurohypophysial system (HNS) release neuropeptides (oxytocin, OT; vasopressin, VP) into the cerebrospinal fluid (CSF) and peripheral plasma from their somatodendrites and axonal terminals, respectively. The vesicles in the HNS fall into two distinct classes, neuropeptide-laden large-dense core vesicles (LDCVs) and microvesicles (MVs). A previous study suggested that a poor Ca2+ sensor protein, synaptotagmin IV (Syt IV), localizes to both classes of vesicles and alters Ca2+-regulated exocytosis of both vesicles. Although MVs are similar to synaptic vesicles (SVs) in size, morphology and molecular composition, their contents and functions in the HNS remain unknown. Here, we examined whether a SV/MV protein, synapsin Ia (Syn Ia), may participate in regulation of neuropeptide release from LDCVs in the HNS of adult male Sprague-Dawley rats. We found that Syn Ia did not colocalize with either oxytocin (OT) or vasopressin (VP)-containing LDCVs in the MCNs or pituitary nerve terminals, suggesting that Syn Ia localizes to SVs/MVs exclusively. To determine the role of SVs/Syn Ia in HNS, we conducted molecular perturbation by in vivo electroporation to unilaterally transfect supraoptic nucleus with various DNA plasmids, including Syt I, Syt IV, wild-type Syn Ia, and its phospho-deficient mutant (Syn Ia-S62A) to abolish SV/MV recruitment to plasma membrane. Sham control was the vector carrying the transfection marker EGFP. To detect the OT/VP changes following various molecular perturbations, CSF and plasma were collected for ELISA measurements before and after in vivo electroporation. Syn Ia significantly increased the OT levels in CSF/plasma, while Syn Ia-S62A did not alter the OT levels in CSF/plasma. Furthermore, cotransfecting Syt IV and Syn Ia also increased the OT levels in CSF/plasma, while this effect was abolished by cotransfecting Syt IV and Syn Ia-S62A. These results suggest that the defective S62 phosphorylation may abolish the Syn Ia-enhanced OT central/peripheral release. Transfection of Syn Ia alone, or Syn Ia together with Syt IV also increased the VP levels in CSF, and this Syn Ia-enhanced VP central release was abolished by defective S62 phosphorylation. In addition, Syn Ia significantly increased the VP levels in plasma, while Syn Ia-S62A slightly decreased the VP levels in plasma. Moreover, the functional Ca2+ sensor protein, Syt I, significantly increased the VP levels in plasma, while Syt IV (harboring a natural mutation in its C2A Ca2+ binding site) did not alter the VP levels in plasma. However, cotransfecting Syt IV and Syn Ia still increased the VP levels in plasma, while this effect was abolished by cotransfecting Syt IV and Syn Ia-S62A. Thus, even though cotransfection with the poor Ca2+ sensor protein Syt IV, Syn Ia was sufficient to enhance VP peripheral release. Taken together, the defective S62 phosphorylation may abolish the Syn Ia-enhanced neuropeptide central/peripheral release. Given that this Syn Ia phosphodeficient mutant blocks SV/MV recruitment, our results suggest that SV/MV recruitment to plasma membrane may facilitate the trafficking and fusion of the other class of vesicles, LDCVs, in the HNS. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T13:29:58Z (GMT). No. of bitstreams: 1 ntu-102-R00b43011-1.pdf: 38039321 bytes, checksum: 9d2375fd7464fc45a8c3f5616f738716 (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | 口試委員審定書 i
致謝 ii 中文摘要 iii Abstract v Abbreviations viii Contents xi Chapter I Introduction 1 1.1 Neurotransmitter release 1 1.2 Large dense-core vesicles and synaptic vesicles 2 1.3 Hypothalamic-Neurohypophysial System 2 1.3.1 Oxytocin and Vasopressin 3 1.3.2 HNS with Central Release and Peripheral release 4 1.3.3 HNS and Microvesicles 5 1.3.4 HNS and MAPK pathway 5 1.4 Synaptotagmin 6 1.4.1 Synaptotagmin I 7 1.4.2 Synaptotagmin IV 8 1.5 Synapsin 10 1.5.1 Synapsin Ia and Phosphorylation 11 1.6 In vivo electroporation 13 1.7 The Purpose of This Study 14 Chapter II Materials and Methods 15 2.1 Animals 15 2.2 DNA Plasmids Construction and Amplifications 15 2.3 Homemade electrodes 18 2.4 Stereotaxic Surgery and In Vivo Electroporation 18 2.5 Heart Perfusion and Tissue fixation 19 2.6 Cryosection 21 2.7 Immunofluorescence chemistry (IFC) 22 2.8 Collection of Plasma and Cerebrospinal Fluid 24 2.9 Enzyme-Linked Immunosorbent Assay (ELISA) 24 2.10 Statistics 26 2.10.1 The OT/ VP and Body Weight Measurement 26 2.10.2 The changes in fluorescence after in vivo electroporation 26 Chapter III Results 27 3.1 Syn I localizes to the somata of SON neurons and posterior pituitary nerve terminals 27 3.2 Syn I is expressed within the somata of SON neurons and posterior pituitary nerve terminals 28 3.3 Syn I colocalizes with the synaptophysin neurons of SON and pituitary nerve terminals 29 3.4 Syn I is expressed in the OT neurons of SON and OT nerve terminals of posterior pituitary 30 3.5 Syt IV is expressed in the OT neurons of SON and the OT nerve terminals of posterior pituitary 31 3.6 Syn I is expressed in the VP neurons of SON and the VP nerve terminals of posterior pituitary 32 3.7 Syt IV is expressed in the VP neurons of SON and the VP nerve terminals of posterior pituitary 33 3.8 Syt IV or Syn Ia is successfully transfected into the SON by in vivo electroporation 33 3.9 Syt IV or Syn Ia is successfully transfected in the posterior pituitary nerve terminals by in vivo electroporation 35 3.10 Syn Ia regulates the OT central release by phosphorylation of S62 37 3.11 Syn Ia regulates the OT peripheral release by phosphorylation of S62 40 3.12 Syn Ia regulates the VP central release by phosphorylation site of S62 44 3.13 Syn Ia regulates the VP peripheral release by phosphorylation of S62 48 3.14 The rat body weight is not changed by overexpressing Syts, wild-type Syn Ia or phosphodeficient Syn Ia 52 Chapter IV Discussion 60 4.1 The role of Syn Ia in regulating LDCVs exocytosis in the HNS 60 4.2 Syn Ia regulates the OT central release, but not significantly affects the VP central release 61 4.3 The role of Syt IV in regulating the neuropeptide release 63 4.4 The OT concentration in plasma after in vivo electroporation is increased in all groups except the Syn Ia* group 64 4.5 Body weight and neuropeptides release 64 4.6 Future Directions 65 Chapter V Conclusion 67 References 68 List of Figures 76 Figure 1. SNARE complex and Synaptotagmin 76 Figure 2. Hypothalamic-Neurohypophysial System 77 Figure 3. Oxytocin and vasopressin are expressed in SON and PVN 78 Figure 4. Large dense-core vesicles and microvesicles in the HNS 79 Figure 5. The Ca2+ binding sites in Synaptotagmin I and Synaptotagmin IV 80 Figure 6. Molecular interaction and phosphorylation of Synapsin I. 81 Figure 7. Homemade electrodes for in vivo electroporation 82 Figure 8. Stereotaxic surgery and in vivo electroporation 83 Figure 9. SON is the target for DNA transfection 84 Figure 10. Syn I immunoreactivity in the somata and axon terminals of SON neurons 85 Figure 11. Syn I localizes to Synaptophysin in the SON neurons and pituitary nerve terminals 87 Figure 12. Syt IV and Syn I localize to OT neurons in the SON neurons and pituitary nerve terminals 88 Figure 13. Syt IV and Syn I localize to VP neurons in the SON neurons and pituitary nerve terminals 90 Figure 14. HA-Syt IV is expressed in the transfected side of SON after in vivo electroporation 92 Figure 15. Myc-Syn Ia is expressed in the transfected side of SON after in vivo electroporation 94 Figure 16. The overexpression levels of Syt IV or Syn Ia in SON after in vivo electroporation 96 Figure 17. Syt IV and Syn I is expressed in the pituitary after in vivo electroporation 97 Figure 18. The changed OT levels in CSF and plasma after transfection 99 Figure 19. The changed VP levels in CSF and plasma after transfection. 101 Figure 20. The body weight in the transfected rats used for measuring OT in CSF and plasma 103 Figure 21. The body weight in the transfected rats used for measuring VP in CSF and plasma 105 Figure 22. The role of Syn Ia or Syt I in regulating central and peripheral release in HNS 107 Figure 23. The role of Syn Ia in regulating LDCVs exocytosis in the HNS 108 List of Tables 109 Table 1. The list of primary antibodies and secondary antibodies 109 Table 2. The list of OT concentration in CSF 110 Table 3. The list of OT concentration in plasma 111 Table 4. The list of VP concentration in CSF 112 Table 5. The list of VP concentration in plasma 113 Table 6. The list of OT changes in CSF and plasma 114 Table 7. Compare groups of OT changes in CSF 115 Table 8. Compare groups of OT changes in plasma 116 Table 9. The list of VP changes in CSF and plasma 117 Table 10. Compare groups of VP changes in CSF 118 Table 11. Compare groups of VP changes in plasma 119 Appendix 120 The 2012 annual meeting of the Society for Neuroscience (New Orleans, Louisiana, U.S.A. 10/13-17/2011): abstract and poster 120 The 2013 College of Life Science Poster (National Taiwan University, Taipei, Taiwan 6/7/2013): abstract and poster 123 | |
dc.language.iso | en | |
dc.title | 在成年雄鼠下視丘-腦下垂體系統中研究Synapsin Ia 調控神經胜肽的釋放所扮演的角色 | zh_TW |
dc.title | The Role of Synapsin Ia in Regulating Neuropeptide Release from the Hypothalamic-Neurohypophysial System of Adult Male Rats | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 周申如(Shen-Ju Chou),徐立中(Li-Chung Hsu),陳示國(Shih-Kuo Chen),盧主欽(Juu-Chin Lu) | |
dc.subject.keyword | synapsin,下視丘-腦下垂體系統,催產素,血管加壓素,large-dense croe vesicle,synaptic vesicle,活體電穿孔, | zh_TW |
dc.subject.keyword | synapsin,hypothalamic-neurohypophysial system,oxytocin,vasopressin,large-dense core vesicles,synaptic vesicles,in vivo electroporation, | en |
dc.relation.page | 124 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2013-07-22 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
顯示於系所單位: | 分子與細胞生物學研究所 |
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