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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 | Hsin-Yo Chen | en |
dc.contributor.author | 陳信祐 | zh_TW |
dc.date.accessioned | 2021-07-11T15:50:19Z | - |
dc.date.available | 2023-08-01 | |
dc.date.copyright | 2018-08-01 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-07-27 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79175 | - |
dc.description.abstract | 在發育初期的雙眼視覺系統中,來自兩眼的視覺訊號會交疊在大腦中兩側的背外側膝狀體(dLGN, dorsal lateral geniculate nucleus)。隨著視覺網路的發育,在dLGN交疊的雙眼視覺訊號會漸漸分開來,而最終雙眼視覺訊號在dLGN會各自佔據一塊互不交疊的區域,而此現象稱作兩眼投射分離現象(eye-specific segregation)。數十年來,研究指出兩眼投射分離現象主要由第二期發育視網膜中的自發性放電現象所調控,而此放電現象簡稱為視網膜波。第二期視網膜波是由星狀無軸突細胞(SACs, starburst amacrine cells)釋放乙醯膽鹼(ACh, acetylcholine)刺激周邊的SACs與視網膜節細胞(RGCs, retinal ganglion cells),而RGCs進而產生神經衝動並將視網膜中的訊息傳遞至中樞神經系統中不同腦區,如本篇研究的dLGN。在本篇研究當中,我們探討Synaptotagmin III第三型鈣離子突觸結合蛋白(Syt3)在發育中視網膜如何調控第二期視網膜波以及下游腦區的視覺迴路。Syt主要存在於突觸前及突觸後神經元,藉其鈣離子結合位點與鈣離子結合,以調控在突觸前神經元神經傳導物質的釋放。而目前已在中樞神經中發現17種Syt,而每種Syt在調控突觸間物質的釋放上有各自的差異性。Syt3在過去實驗室研究中發現,在出生後大鼠的RGC中,Syt3在第二期視網膜中短暫地大量表現於調控兩眼投射分離現象的關鍵時期,因此我們假設RGC中的Syt3在調控第二期視網膜波及下游的視覺迴路上扮演一重要角色。因此我們利用點突變、體外電穿孔轉染、活體電穿孔轉染、活細胞鈣離子顯像技術、冷凍切片、螢光染色等技術來證明此假設。
首先,我們利用體外電穿孔轉染在RGC中大量表現正常的Syt3及兩個鈣離子結合蛋白各自突變的Syt3 變異株(Syt3-C2A*及Syt3-C2B*),我們發現正常的Syt3與其變異株在調控第二期視網膜波的時空特性上,有相似的結果。其二,我們利用活體電穿孔轉染在RGC中大量表現正常的Syt3以及會轉錄出反序列且互補的Syt3片段以干擾及降低原生Syt3的表現。我們發現正常的Syt3在調控第二期視網膜波的時空特性上並沒有顯著的差異;反之,降低原生Syt3的表現會使第二期視網膜波中鈣離子流入的持續時間與強度下降。其三,我們接著探討由Syt3影響的第二期視網膜波如何影響下游的視覺迴路形成。結果顯示,在RGC中降低原生Syt3的表現會改變大腦兩側dLGN中的兩眼投射分離現象。其四,從過去實驗數據推論麩胺酸在調控第二期視網膜波可能扮演重要角色。因此我們間隔性施打離子型麩胺酸受體拮抗劑(iGluR Antagonists)以影響第二期視網膜波及觀察dLGN中的兩眼投射分離現象。結果發現在第二期視網膜波中,受影響的麩胺酸訊息傳遞會改變在大腦同側dLGN中的兩眼投射分離現象。從上述結果,我們推論Syt3在調控第二期視網膜波及兩眼投射分離現象上扮演重要角色;且在視網膜中,正常的麩胺酸訊息傳遞對兩眼投射分離現象也相當重要。 | zh_TW |
dc.description.abstract | In the developing binocular visual system, axon terminals projecting from two eyes are initially evenly innervated in a region of thalamus, i.e., the dorsal lateral geniculate nucleus (dLGN). During a developmental critical period (P4-P8 in rodent), the innervation of unwanted terminals in the dLGN is further refined, leading to the eye-specific segregation of the dLGN. Previous studies found that the eye-specific segregation of the dLGN requires patterned spontaneous activity in the developing retina, termed stage II retinal waves, propagating through developing starburst amacrine cells (SACs) and retinal ganglion cells (RGCs) to the visual cortex. We previously found synaptotagmin III (Syt3), a Ca2+ sensor, is transiently overexpressed in RGCs during the developmental critical period of eye-specific segregation and stage II retinal waves. However, how Syt3 in developing RGCs regulates stage II retinal waves and the eye-specific segregation of the dLGN remains unclear. To study the role of the Ca2+-binding domains (C2A and C2B) of Syt3 in regulating stage II retinal waves, we overexpressed Syt3-C2A* or Syt3-C2B* in RGCs and found that both mutants did not significantly alter stage II retinal waves compared to the wild-type Syt3, suggesting that both C2 domains of Syt3 in RGCs weigh similarly in regulating retinal waves. Second, to determine the role of Syt3 in RGCs in regulating the eye-specific segregation of the dLGN, we eliminated the expression of Syt3 by overexpressing the anti-sense Syt3 in RGCs using in vivo electroporation at P3. At P8, we examined the eye-specific segregation of the dLGN using axonal tracing and immunofluorescence staining. We found that the in vivo elimination of Syt3 in RGCs altered the eye-specific segregation of the dLGN and dampened stage II retinal waves in terms of wave duration and amplitude. These data suggest that Syt3 in developing RGCs is crucial for the eye-specific segregation of the dLGN. Consistently, the blockade of glutamate transmission in developing retinas regulates the eye-specific segregation of the dLGN, implying that glutamate release from RGCs within the retina is required for the correct eye-specific segregation of the dLGN. Together, our results support that Syt3 in developing RGCs can modulate patterned spontaneous activity in the retina and visual circuit refinement. | en |
dc.description.provenance | Made available in DSpace on 2021-07-11T15:50:19Z (GMT). No. of bitstreams: 1 ntu-107-R05b43006-1.pdf: 12404465 bytes, checksum: 6a40d59bb8fc56ce690215b304c33e5e (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 口試委員審定書 i
Acknowledgements ii 中文摘要 iii Abstract v Abbreviations vii Content x Chapter I - Introduction 1 1.1 Visual System and Image-forming Brain Areas 1 1.2 Anatomy of the Retina 2 1.3 Retinal Development 3 1.4 Synaptic Transmission and Exocytosis 4 1.5 Synaptotagmin 5 1.6 Synaptotagmin III 6 1.7 Patterned Spontaneous Activity 7 1.8 Properties of Retinal Waves 7 1.9 Retinal Waves Instruct Maturation of Visual Circuits 8 1.10 Previous works for the role of Syt3 in regulating stage II retinal waves and visual circuits 11 1.11 Aims in this study 12 Chapter II - Materials and Methods 14 2.1 Plasmids 14 2.2 Animals 15 2.3 Dissection of Retinas 16 2.4 Primary Culture of Retinal Explants 17 2.5 Ex vivo electroporation 17 2.6 In vivo electroporation and intraocular drug administration 18 2.7 Anterograde axonal labeling 19 2.8 Live Ca2+ Imaging 20 2.9 Analysis of Spontaneous Ca2+ Transients – Temporal Properties 22 2.10 Analysis of Spontaneous Ca2+ Transients – Spatial Properties 22 2.11 Transcardiac Perfusion and Sample Fixation 24 2.12 Cryosection of Eyeballs, Brains, and Optic nerves 24 2.13 Antibodies 25 2.14 Immunofluorescence and acquisition of fluorescence images 26 2.15 Quantification of territories and eye-specific segregation in the dLGN, and the signal intensity of the cross sections of the optic nerves 27 2.16 Statistics 29 Chapter III - Results 30 3.1 The C2A and C2B domains of Syt3 weigh similarly in regulating temporal properties of stage II retinal waves. 30 3.2 The C2A and C2B domains of Syt3 weigh similarly in regulating spatial properties of stage II retinal waves. 32 3.3 Plasmids of interest can be overexpressed in developing RGCs by in vivo electroporation. 33 3.4 Reducing the level of Syt3 in RGCs during stage II retinal waves shrinks the ipsilateral territory in the transfected-side and expands the ipsilateral territory in the untransfected-side dLGN. 36 3.5 Reducing the level of Syt3 in RGCs during stage II retinal waves increases eye-specific segregation in the transfected-side and decreases in the untransfected-side dLGN. 38 3.6 Reducing the level of Syt3 in a limited number of RGCs affects stage II retinal waves in aspects of the duration and amplitude, while elevating the level of Syt3 does not. 39 3.7 Reducing or elevating the level of Syt3 in a fraction of RGCs does not affect spatial properties of stage II retinal waves. 41 3.8 Disturbing glutamatergic transmission in developing retinas expands the ipsilateral territory in the injected-side dLGN. 42 3.9 Disturbing glutamatergic transmission in developing retinas reduces eye-specific segregation in the injected-side dLGN. 44 3.10 Disturbing glutamatergic transmission in developing retinas does not affect the temporal properties of stage II retinal waves. 44 3.11 Disturbing glutamatergic transmission in developing retinas does not affect spatial properties of stage II retinal waves. 45 3.12 Disturbing glutamatergic transmission increases the expression of GluA2 subunits in developing retinas. 46 Chapter IV - Discussion 48 4.1 Ca2+ binding to the C2A and C2B of Syt3 in RGCs weigh similarly in regulating stage II retinal waves. 49 4.2 The manipulated level of Syt3 in ex vivo or in vivo RGCs affects stage II retinal waves. 50 4.3 The manipulated level of Syt3 in RGCs in one retina disrupts the balance of Syt3 expression and the dynamics of retinal waves in the same pup. 53 4.4 Syt3-modulated stage II retinal waves establish correct neural circuits in the dLGN. 54 4.5 Glutamatergic transmission in developing retinas maintain the ipsilateral projections to the dLGN. 57 Chapter V - Conclusion 60 References 61 Figure 1. The image-forming visual pathway in rodents . 69 Figure 2. Anatomy of vertebrate retinas. 70 Figure 3. Three stages of retinal waves during development. 71 Figure 4. Synaptotagmin in neuronal exocytosis and its Ca2+ binding domains. 72 Figure 5. Eye-specific segregation in the dorsal lateral geniculate nucleus (dLGN). 74 Figure 6. Synaptotagmin III’s expression during stage II retinal waves. 75 Figure 7. Analysis of eye-specific segregation in the dLGN 77 Figure 8. The frequency and interval of spontaneous Ca2+ transients remain similar by overexpressing Syt3, Syt3-C2A*, or Syt3-C2B* in RGCs. 78 Figure 9. The duration and amplitude of spontaneous Ca2+ transients remain similar by overexpressing Syt3, Syt3-C2A*, or Syt3-C2B* in RGCs. 80 Figure 10. The spike time tiling coefficients (STTC) of pairs of spontaneous Ca2+ transients remain unchanged in retinal explants overexpressing Syt3-C2A* and Syt3-C2B* in RGCs compared to Syt3. 83 Figure 11. The correlated activity of spontaneous Ca2+ transients remains unchanged in retinal explants overexpressing Syt3-C2A* and Syt3-C2B* in RGCs compared to Syt3. 84 Figure 12. The schematic flowchart to study retinal waves and retinogeniculate projections via in vivo electroporation. 86 Figure 13. HA-tagged Syt3 can be locally expressed and detected at the nasal side of the whole-mount retina after in vivo electroporation. 88 Figure 14. The reduced level of Syt3 in a fraction of RGCs can be detected around the site of injection after in vivo overexpression of anti-sense Syt3. 91 Figure 15. The elevated or reduced level of Syt3 is discernible in RGC axons after in vivo expression of plasmids of interest. 93 Figure 16. Reducing the level of Syt3 in a fraction of RGCs during development shrinks the ipsilateral area in the transfected-side but expands the ipsilateral area in the untransfected-side dLGN. 95 Figure 17. Reducing the level of Syt3 in a fraction of RGCs during development increases eye-specific segregation in the transfected-side but decreases eye-specific segregation in the untransfected-side dLGN. 97 Figure 18. The interval and frequency of spontaneous Ca2+ transients in stage II retinal waves remain unchanged by overexpressing the anti-sense Syt3 sequence in RGCs. 98 Figure 19. Reducing levels of Syt3 in a limited number of RGCs dampens the duration and amplitude of spontaneous Ca2+ transients in stage II retinal waves. 100 Figure 20. The spike time tiling coefficient (STTC) remains unchanged by reducing or elevating Syt3 levels in a fraction of RGCs. 103 Figure 21. The correlated activity remains unchanged by reducing or elevating Syt3 levels in a fraction of RGCs. 106 Figure 22. The schematic flowchart to study retinal waves and retinal projections via intraocular drug administration. 108 Figure 23. Continual intraocular injections of iGluR antagonists during development expand the ipsilateral area in the injected-side dLGN. 111 Figure 24. Continual intraocular injections of iGluR antagonists during development reduce eye-specific segregation in the injected-side dLGN. 113 Figure 25. Continual intraocular injections of iGluR antagonists during development preserve the integrity of the retina and induce the higher expression of AMPA receptor subunit 2 (GluA2) in the GCL in the whole-mount retinas. 114 Figure 26. Continual intraocular injections of iGluR antagonists during development preserve the integrity of the retina and induce the higher expression of AMPA receptor subunit 2 (GluA2) in the GCL in the cross sections of the retinas. 117 Figure 27. The interval and frequency of spontaneous Ca2+ transients in stage II retinal waves remain unchanged after ~24 hr post injection of iGluR antagonists. 118 Figure 28. The duration and amplitude of spontaneous Ca2+ transients in stage II retinal waves remain unchanged after ~24 hr post injection of iGluR antagonists. 120 Figure 29. The spike time tiling coefficient (STTC) remains unchanged after ~24 hr post injection of iGluR antagonists 122 Figure 30. The correlated activity remains unchanged after ~24 hr post injection of iGluR antagonists 124 Figure 31. Summary of results linking retinal waves to eye-specific segregation in the dLGN after in vivo electroporation or intraocular injections of drugs. 127 Figure 32. Summary of the altered wave properties and eye-specific segregation in the dLGN after in vivo electroporation or intraocular injections of drugs. 128 Figure 33. Syt3 in RGCs plays an important role in regulating stage II retinal waves and visual circuit development. 129 Appendix 130 Society of Neuroscience (SfN) 2017 Abstract and Poster 131 Institute of Molecular and Cellular Biology (IMCB) 2018 Abstract and Poster 134 | |
dc.language.iso | en | |
dc.title | 第三型突觸連結蛋白調控模式化自發性放電現象與視覺網路發育的機制 | zh_TW |
dc.title | The Mechanism of Synaptotagmin III in Regulating Patterned Spontaneous Activity and Visual Circuit Development | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 盧主欽(Juu-Chin Lu),周申如(Shen-Ju Chou),焦傳金(Chuan-Chin Chiao) | |
dc.subject.keyword | 第三型突觸鈣離子結合蛋白,第二期視網膜波,麩胺酸突觸傳導,兩眼投射分離現象,活細胞鈣離子顯像技術,活體電穿孔轉染, | zh_TW |
dc.subject.keyword | Synaptotagmin III,Stage II retinal waves,Glutamate Release,Eye-Specific Segregation,Live Ca2+ Imaging,In Vivo Electroporation, | en |
dc.relation.page | 136 | |
dc.identifier.doi | 10.6342/NTU201802082 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2018-07-30 | |
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 |
dc.date.embargo-lift | 2023-08-01 | - |
顯示於系所單位: | 分子與細胞生物學研究所 |
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