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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王致恬 | |
| dc.contributor.author | Yi-Wen Lin | en |
| dc.contributor.author | 林怡彣 | zh_TW |
| dc.date.accessioned | 2021-07-11T15:48:04Z | - |
| dc.date.available | 2023-08-06 | |
| dc.date.copyright | 2018-08-06 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-08-02 | |
| dc.identifier.citation | Arroyo, D. A., Kirkby, L. A., & Feller, M. B. (2016). Retinal Waves Modulate an Intraretinal Circuit of Intrinsically Photosensitive Retinal Ganglion Cells. J Neurosci, 36(26), 6892-6905. doi:10.1523/jneurosci.0572-16.2016
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79149 | - |
| dc.description.abstract | 視覺迴路的修飾與成熟倚賴視網膜的一種模式化、規律化的自發性活動，稱作視網膜波。在老鼠出生後的第一周，視網膜波的產生是由於一群突觸前神經元─星狀無軸突細胞釋放神經傳導物(如：乙醯膽鹼和γ-氨基丁酸)至四周的星狀無軸突細胞和突觸後神經元─神經節細胞，使得這些細胞活化而產生視網膜上的細胞活動，如水波一般的散開。Synaptotagmin I (Syt I) 參與在神經傳導物釋放的胞吐作用中，是一種鈣離子偵測蛋白。而我們之前的研究發現，突觸後神經元─神經節細胞的 Syt I 在調控視網膜波中，扮演重要的角色，這顯示神經節細胞釋放的神經傳導物也會影響視網膜波的特性。然而，目前還不知道，突觸後的 Syt I 調控視網膜波的現象，是否只需特定種類神經節細胞的參與其中。在本研究中，我們提出了假說，假定此現象只需要感光視神經節細胞中的 Syt I 參與。為了驗證我們的假說，我們進行了分子生物技術的干擾、免疫螢光染色的實驗，還有鈣離子影像技術等實驗。
首先，為了了解 Syt I 在視網膜中扮演的功能與角色，我們將 Syt I 上和鈣離子結合有關的重要區域 C2A 與 C2B 進行點突變 (Syt I-C2AB*)，以減弱這兩個區域與蛋白質結合的能力。我們發現將 Syt I-C2AB* 過度表現在神經節細胞上，和表現野生型的 Syt I 的組別比較，視網膜波的特性並沒有改變。為了證明感光視神經節細胞參與在突觸後的 Syt I 調控視網膜波的現象，我們利用免疫螢光染色，證明初生老鼠的感光視神經節細胞會表現 Syt I，而我們也發現，先前研究用來專一表現 Syt I 在神經節細胞中的啟動子也可以標的到感光視神經節細胞上，證實感光視神經節細胞在調控視網膜波中佔有一席之地。接著我們將野生型的 Syt I 和Syt I-C2AB* 過度表現在感光視神經節細胞上，結果發現和對照組相比，這兩個實驗組都可以使視網膜波的頻率顯著提升，但對其他波的特性沒有影響。這個結果，顯示感光視神經節細胞中的 Syt I 即足以調控視網膜波的頻率，證實了感光視神經節細胞在突觸後 Syt I 調控視網膜波的現象中，扮演重要的角色。 | zh_TW |
| dc.description.abstract | The maturation of visual circuits requires the retinas bursting patterned spontaneous activities, named retinal waves, for visual circuit refinement. During the first week of postnatal development in rodent, retinal waves are initiated by calcium-mediated exocytosis to release acetylcholine and γ-aminobutyric acid from the starburst amacrine cells (SACs) onto the neighbor SACs and retinal ganglion cells (RGCs). Our previous studies found that in retinal ganglion cells (RGCs), synaptotagmin I (Syt I), a calcium sensor important for presynaptic release, affects the spatiotemporal properties of retinal waves, indicating that the postsynaptic RGCs release also mediates retinal waves. However, which subtypes of RGCs mediate retinal waves via Syt I remains unknown. Here, we hypothesized that the melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) may be the potential candidate RGC subtype. To provide the evidences for the hypothesis, we explored the functional role of Syt I in ipRGCs, by combining molecular perturbation, immunofluorescence staining, and live Ca2+ imaging.
We established a model to examine the functional role of Syt I, by weakening Ca2+ binding to the C2A and C2B domains of Syt I (designated Syt I-C2AB*). We found that overexpressing Syt I-C2AB* did not affect retinal waves compare to Syt I. To demonstrate the expression of Syt I in ipRGCs, immunofluorescence staining was conducted on retinal cross-sections and whole-mount retinal explants. The results showed that ipRGCs expressed Syt I during postnatal development and that ipRGCs can be targeted by the Brn3b promoter, the promoter used to target Syt I in RGCs in previous studies showing Syt I’s role in regulating retinal waves. Furthermore, we verified the specificity of the Opn4 promoter to target gene expression in ipRGCs by immunofluorescence staining. Last but not least, live Ca2+ imaging showed that the overexpression of Syt I or Syt I-C2AB* in ipRGCs affected wave frequency and interval, but not wave size and spatial properties. Our results showed that Syt I in ipRGCs is enough to regulate retinal waves frequency, suggesting that ipRGCs are involved in Syt I’s regulation of retinal waves. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T15:48:04Z (GMT). No. of bitstreams: 1 ntu-107-R05B43017-1.pdf: 12992482 bytes, checksum: 6fd67b814a2f80619240939b2efb9640 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 口試委員審定書 i
致謝 ii 中文摘要 iii Abstract v Abbreviations vii Chapter I Introduction 1 1.1 The development of nervous system 1 1.2 Organization of the visual system 2 1.3 Structures and physiology of retinas: RGC subtypes 3 1.4 The spontaneous patterned activities in developing visual system 5 1.5 Mechanisms to mediate stage II retinal waves 7 1.6 Calcium-regulated exocytosis 9 1.7 Synaptotagmin I 10 1.8 Syt I in RGCs: Retrograde signal to regulate stage II retinal waves 12 1.9 Hypothesis 13 1.10 Aims of this study 16 Chapter II Materials and Methods 18 2.1 Subcloning and site-directed mutagenesis 18 2.2 Animals 25 2.3 Whole-mount retinal preparation 26 2.4 Primary retinal explant culture 27 2.5 Ex vivo electroporation 27 2.6 Antibodies 28 2.7 Immunofluorescence staining 29 2.8 Live calcium imaging 30 2.9 Data analysis of spontaneous calcium transients 32 2.10 Statistical analysis 34 Chapter III Results 35 3.1 Weakened Ca2+ binding to both C2A and C2B domains of Syt I does not affect retinal wave frequency. 35 3.2 Overexpressing Syt I-C2AB* in RGCs does not change the duration and amplitude of Ca2+ transients. 37 3.3 The spatial correlation of Ca2+ transients was not affected by overexpressing Syt I-C2AB* in RGCs. 38 3.4 Syt I is expressed in neonatal ipRGCs. 40 3.5 The Brn3b promoter can target gene expression partially to ipRGCs. 41 3.6 The Opn4 promoter can target gene expression specifically to ipRGCs. 42 3.7 The retinal wave frequency is increased by overexpressing Syt I or Syt I-C2AB* in ipRGCs. 43 3.8 The retinal wave duration and amplitude are not affected by overexpressing Syt I or Syt I-C2AB* in ipRGCs. 44 3.9 The spatial correlation of Ca2+ transients is slightly reduced by overexpressing Syt I in ipRGCs. 45 Chapter IV Discussion. 48 4.1 Syt I’s role in regulating stage II retinal waves. 48 4.2 The role of ipRGCs in regulating stage II retinal waves. 50 4.3 Significance and future work. 53 Chapter V Conclusion. 55 References 56 List of Figures Figures for Introduction Figure 1. An overview of the retinofugal pathway. 66 Figure 2. Schemata of the cross section of an eyeball and a mature retina. 67 Figure 3. Figure 3. Illustration and mechanisms of retinal waves. 69 Figure 4. The mechanism of vesicle exocytosis and the structure of Synaptotagmin I 71 Figure 5. Syt I is expressed during developmental stages in the rat retina. 73 Figure 6. Syt I in RGCs regulates stage II retinal waves. 75 Figure 7. Bath applying glutamate occludes the effect of Syt I-C2A on wave frequency. 77 Figures for Results Figure 8. Recordings of spontaneous Ca2+ transients after exo vivo retinal transfection. 79 Figure 9. Overexpressing Syt I in RGCs increases the frequency of spontaneous Ca2+ transients. 81 Figure 10. Overexpressing Syt I or Syt I-C2AB* in RGCs does not affect the duration or amplitude of spontaneous Ca2+ transients. 83 Figure 11. Pairwise correlation index (C.I.) is decreased by overexpressing Syt I in RGCs. 85 Figure 12. Overexpressing Syt I in RGCs slightly affects the waves spatial properties. 86 Figure 13. Overexpressing Syt I-C2AB* in RGCs slightly affects the correlation of the activities of the cells selected in one imaged region. 87 Figure 14. Localization of Syt I and ipRGCs in the cross-sections of the P1 rat retina. 89 Figure 15. Localization of Syt I and ipRGCs in the cross-sections of the P7 rat retina. 90 Figure 16. The HA-Syt I driven by pBrn3b expresses partially around ipRGCs. 91 Figure 17. The DNA sequence of the Opn4 promoter used in this study. 92 Figure 18. The HA-Syt I driven by Opn4 expresses surrounding ipRGCs. 94 Figure 19. Overexpressing Syt I or Syt I-C2AB* in ipRGCs increases the frequency of spontaneous Ca2+ transients. 96 Figure 20. Overexpressing Syt I or Syt I-C2AB* in ipRGCs does not affect the duration or amplitude of spontaneous Ca2+ transients. 96 Figure 19. Overexpressing Syt I or Syt I-C2AB* in ipRGCs increases the frequency of spontaneous Ca2+ transients. 98 Figure 21. Pairwise correlation index (C.I.) is decreased by overexpressing Syt I or Syt I-C2AB* in ipRGCs. 100 Figure 22. Overexpressing Syt I or Syt I-C2AB* in ipRGCs affects the waves spatial properties. 101 Figure 23. Overexpressing Syt or Syt I-C2AB* in ipRGCs does not affect the correlation of the activities of the cells selected in one imaged region. 102 List of Tables Table 1. The list of primers. 104 Table 2. The list of antibodies used in this study. 106 Table 3. The values of C.I. for each 50-μm bins in transfected retinal explants. 107 Table 4. The values of STTC for each 50-μm bins in transfected retinal explants. 100 Table 5. The specific statistical method for each comparison of the C.I. values or STTC values. 101 Table 6. The specific-statistical method for each comparison of the distributions of cumulative probabilities for the fraction of cells. 102 | |
| dc.language.iso | en | |
| dc.title | 在發育過程中感光神經節細胞的 Synaptotagmin I 調控視網膜波的角色 | zh_TW |
| dc.title | The role of ipRGCs’ Synaptotagmin I in regulating
retinal waves during development | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 盧主欽,焦傳金 | |
| dc.subject.keyword | 視網膜波,Synaptotagmin I,感光視神經節細胞,鈣離子影像技術,自發性活動,神經發育, | zh_TW |
| dc.subject.keyword | retinal waves,Synaptotagmin I,ipRGCs,live-calcium imaging,spontaneous activities,neural circuit refinement, | en |
| dc.relation.page | 116 | |
| dc.identifier.doi | 10.6342/NTU201802299 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-08-02 | |
| 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 | 2023-08-06 | - |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
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