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請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72457
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.authorSheng-Ping Hsuen
dc.contributor.author徐聖平zh_TW
dc.date.accessioned2021-06-17T06:59:26Z-
dc.date.available2024-08-07
dc.date.copyright2019-08-07
dc.date.issued2019
dc.date.submitted2019-08-05
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dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72457-
dc.description.abstract在發育的關鍵時期,星狀無軸突細胞(SACs, starburst amacrine cells)釋放乙烯膽鹼而產生第二期視網膜波在發育的視網膜中傳遞,其中視網膜節細胞(RGCs, retinal ganglion cells)會將視網膜波的影響傳遞至我們的大腦中,去調控視覺迴路的發育。我們實驗室先前的研究指出除了星狀無軸突釋放乙烯膽鹼之外,視網膜節細胞也會藉由釋放麩胺酸去調控視網膜波的特性,因此影響視覺迴路的發育,像是在背外側膝狀體(dLGN, dorsal lateral geniculate nucleus) 的兩眼投射分離現象(eye-specific segregation),然而我們還不清楚麩胺酸如何調控第二期視網膜波。在本篇研究中,我們以不同層面去研究麩胺酸如何影響第二期視網膜波。首先,為了確認麩胺酸也會在體內調控第二期視網膜波,我們施打離子性麩胺酸受體拮抗劑(iGluR antagonists)至仔鼠玻璃體去抑制麩胺酸的訊號傳遞後,利用活體鈣離子顯像技術來觀察視網膜波的變化,發現打藥之後視網膜的頻率和細胞同步性有顯著地降低,表示麩胺酸會調控第二期視網膜波的時空特性。另外,在抑制麩胺酸的訊號之後,我們發現其中一個麩胺酸受體:AMPA 受體的次單元(GluA2)的蛋白表現量有顯著的上升,表示抑制麩胺酸訊號傳遞會影響視網膜的基因表現量。第二,為了知道GluA2在發育中視網膜中的分布,我們藉由免疫螢光染色去觀察不同出生日期的仔鼠視網膜,發現GluA2大部分表現在內叢狀層 (IPL, inner plexiform layer) 和神經節細胞層 (GCL, ganglion cell layer)。接著,我們想知道麩胺酸如何藉由自分泌訊號傳遞去影響視網膜波,我們藉由降低RGCs中GluA2表現量,然後進行活體鈣離子顯像技術去觀察視網膜波的變化,發現降低RGCs中GluA2表現量後,視網膜波的頻率和細胞同步性有顯著地上升,表示麩胺酸會藉由自分泌訊號傳遞去調控第二期視網膜波的時空特性。最後,為了知道GluA2的動態平衡在第二期視網膜波中是否會因為不同刺激而有變化,所以我們藉由酸鹼敏感綠螢光蛋白(pHluorin)和GluA2的融合蛋白去觀察發育時期視網膜不同刺激下GluA2的動態平衡的改變,若使用腺甘酸受體促進劑去增加第二期視網膜波的活性,亦會促進GluA2的胞吞作用;若加入更多麩胺酸或是iGluR antagonists,傾向GluA2的胞吐作用。這些結果表示GluA2的動態平衡會因為視網膜波的活性而有改變。綜合以上結果,我們發現麩胺酸會藉由RGCs上含GluA2的AMPA受體來調控視網膜波。另外,GluA2在細胞膜上的動態平衡也可能是發育時期影響視網膜波的重要的調控機制。zh_TW
dc.description.abstractDuring a critical period of visual circuit refinement, stage II retinal waves are initiated by the release from starburst amacrine cells (SACs), propagating throughout the layer containing retinal ganglion cells (RGCs). We previously found that RGCs may release glutamate diffusely in the inner plexiform layer (IPL) and ganglion cell layer (GCL), thus modulating stage II retinal waves and further regulating the eye-specific segregation of the dorsal lateral geniculate nucleus (dLGN) in thalamus. However, the detail in glutamate regulation of stage II retinal waves remains unclear. First, to investigate how glutamate transmission affects retinal waves in vivo, we performed the intraocular injection of iGluR antagonists in rat pups. We found that wave frequency and spike time tiling coefficient (STTC) across distance were downregulated by the intraocular blockade of iGluR in vivo, suggesting that glutamate transmission modulates wave properties in vivo. Additionally, we found that the expression level of AMPA subunit 2 (GluA2) was increased upon the intraocular application of iGluR antagonists in vivo, suggesting that the inhibition of glutamate transmission may lead to the alterations in gene expression during stage II retinal waves. Second, to determine the presence of the glutamate receptor in developing retinas, we performed immunofluorescence staining and found that the GluA2 was mainly expressed in the IPL and GCL. Further, to investigate the role of glutamate autocrine regulation via RGCs, we transfected the anti-sense GLUA2 to specifically knockdown the GluA2 expression in RGCs. We found that wave frequency and STTC across distance were upregulated by GluA2 depletion, suggesting that glutamate receptors may mediate the autocrine regulation via RGCs, thus regulating the wave frequency and firing correlation. Third, to examine whether the GluA2 dynamics on plasma membrane can be changed upon different stimuli, we transfected the developing retinas with the optical reporter of GluA2 trafficking (i.e., the fusion protein of pHluorin and GluA2), we found that GluA2 internalization was promoted by application of CGS 21680, a selective agonist of adenosine A2A receptor shown to increase wave frequency via SACs. Furthermore, we found that GluA2 externalization was promoted by the minute level of glutamate or by iGluR antagonists. Our results suggest that wave activity may regulate the dynamics of GluA2 trafficking, and the homeostasis of glutamate transmission is maintained by a critical level of transmitter-receptor interaction during stage II retinal waves. Together, our data suggest that glutamate transmission during the stage II wave period may act via RGCs, through AMPA receptors. The dynamics of AMPA receptors in RGCs may play an important role in modulating stage II retinal waves during retinal circuit refinement.en
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dc.description.tableofcontents國立臺灣大學碩士學位論文口試委員會審定書 i
致謝 ii
中文摘要 iii
Abstract v
Abbreviations vii
Chapter I: Introduction 1
1.1 The structure of the mature vertebrate retina and visual pathway 1
1.2 Neural refinement and retinal waves 2
1.3 The properties of stage II retinal waves and visual circuit formation 4
1.4 Calcium-dependent exocytosis and Synaptotagmins 6
1.5 Previous studies in our lab 8
1.6 Glutamate release and glutamate receptors: 10
1.7 Glutamate signaling in LTP/LTD 12
1.8 The expression of iGluRs in developing retinas 14
1.9 Objective of this study 14
Chapter II: Materials and Methods 17
2.1 Plasmid information 17
2.2 Animals 18
2.3 Retinal dissection 18
2.4 Ex vivo transfection 19
2.5 Dissociated retinal neurons 20
2.6 Immunofluorescence staining and antibodies 21
2.7 Image acquisition and quantification 25
2.8 Live Ca2+ imaging 26
2.9 Intraocular injection 29
2.10 RNA extraction 29
2.11. Quantitative reverse transcription PCR (RT-qPCR) 30
2.12 Live GluA2 imaging 31
2.13 Statistics 33
Chapter III: Results 34
3.1. Temporal and spatial properties of retinal waves are not changed after repetitive application of iGluR antagonists. 34
3.2. Short-term blockade of glutamate transmission regulates the wave interval and frequency, but not the wave duration and amplitude. 36
3.3. Short-term blockade of glutamate transmission regulates the affect firing correlation of retinal waves. 38
3.4. Repetitive in vivo blockade of glutamate transmission increase the expression level of GluA2 protein in the GCL. 38
3.5. Repetitive in vivo blockade of glutamate transmission in one eye regulates the retinal gene expression in the other eye. 40
3.6 Repetitive in vivo blockade of glutamate transmission promotes phosphorylation of CREB. 42
3.7. GluA2 is expressed in the developing inner retinas and localized to both SACs and RGCs. 43
3.8 The 50% knockdown efficiency for GluA2 in RGCs after ex vivo transfection 45
3.9. Autocrine glutamate transmission may enhance the wave frequency but diminish the wave interval. 46
3.10. Autocrine glutamate transmission regulates the firing correlation of retinal waves. 47
3.11. The CMV promoter-driven pHluorin-GluA2 is expressed in both SACs and RGCs, with the enhanced intensity upon inhibiting vesicle acidification. 47
3.12 The increased wave activity promotes GluA2 internalization. 49
3.13 The 5 nM extracellular glutamate tends to promote GluA2 externalization. 49
3.14 Blockade of glutamate transmission tends to promote GluA2 externalization. 51
Chapter IV: Discussion 53
4.1. Glutamate transmission regulates stage II retinal wave in vivo. 55
4.2. Inhibition of glutamate transmission affects binocular gene expression during the period of stage II retinal waves. 56
4.3. The interaction between wave activities and GluA2 expression. 58
4.4 The future work in the detailed mechanisms of glutamate regulation in stage II retinal waves 61
Chapter V: Conclusion 63
References 64
Figure 1. The structure of mature rodent retinas. 70
Figure 2. The visual pathway in the mature rodent brain. 71
Figure 3. Stage II retinal waves initiate from ACh release from SACs, mediated by Ca2+-dependent exocytosis. 73
Figure 4. Previous studies suggested that RGCs release glutamate and regulate stage II retinal waves, further affecting eye-specific segregation. 74
Figure 5. The illustration of intraocular injection of iGluR antagonists and flow chart of Aim I 77
Figure 6. Sample traces recorded from Ca2+ imaging after repetitive application of iGluR antagonists. 78
Figure 7. Repetitive intraocular injection of iGluR antagonists does not affect the wave interval and frequency. 79
Figure 8. Repetitive intraocular injection of iGluR antagonists does not affect the wave duration and amplitude. 81
Figure 9. Repetitive intraocular injection of iGluR antagonists does not affect the spike time tailing coefficient (STTC) of retinal waves. 84
Figure 10. Repetitive intraocular injection of iGluR antagonists does not affect correlated activity of retinal waves. 86
Figure 11. Sample traces recorded from Ca2+ imaging after single intraocular injection of iGluR antagonists. 87
Figure 12. Single intraocular injection of iGluR antagonists downregulates the frequency but upregulates the interval of Ca2+ waves. 88
Figure 13. Single intraocular injection of iGluR antagonists does not affect the duration or amplitude of Ca2+ waves. 90
Figure 14. Single intraocular injection of iGluR antagonists downregulates STTC but not correlation activity of Ca2+ waves. 92
Figure 15. Repetitive intraocular injection of iGluR antagonists increases GluA2 expression in the developing GCL. 95
Figure 16. Repetitive intraocular injection of iGluR antagonists does not apparently affect GluA2 expression in the developing INL. 97
Figure 17. Repetitive intraocular injection of iGluR antagonists promoted the GluA2 expression in both SACs and RGCs of the GCL. 98
Figure 18. Repetitive intraocular injection of iGluR antagonists differentially regulates the mRNA levels of Nr1, Syt I, Syt III, or Syt IV in the iGluR antagonist-uninjected retinas. 100
Figure 19. Repetitive intraocular injection of iGluR antagonists increases the CREB phosphorylation in the developing retinas. 103
Figure 20. The AMPA subunit GluA2 is expressed in developing rat retinas, especially in the IPL and the GCL. 105
Figure 21. SACs and RGCs express GluA2 at P2 and P6. 106
Figure 22. The flow chart and constructs used in Aim II 107
Figure 23. Knockdown of GluA2 in developing RGCs by ex vivo transfection. 108
Figure 24. Sample traces recorded from Ca2+ imaging after ex vivo transfection of Ctrl and AS GluA2. 110
Figure 25. Knockdown of GluA2 in developing RGCs upregulates the frequency but downregulates the in interval of Ca2+ waves. 111
Figure 26. Knockdown of GluA2 in developing RGCs does not affect the duration or amplitude of Ca2+ waves. 113
Figure 27. Knockdown of GluA2 in developing RGCs upregulates the firing correlation of Ca2+ waves. 115
Figure 28. The flow chart of pHluorin-GluA2 for real-time GluA2 imaging. 118
Figure 29. The data analysis of GluA2 imaging. 120
Figure 30. pHluorin-GluR2 is expressed in both RGCs and SACs after ex vivo transfection. 121
Figure 31. Bath application of CGS promoted GluA2 endocytosis in the cells of GCL. 122
Figure 32. Bath application of 100 μM glutamate biphasically affects GluA2 dynamic in the cells of the GCL. 124
Figure 33. Bath application of 5 nM glutamate tends to promote GluA2 exocytosis in the cells of the GCL. 126
Figure 34. Bath application of iGluR antagonists tends to promote GluA2 exocytosis in the cells of the GCL. 128
Figure 35. The summary of glutamate-transmission regulation and interaction between wave activities and GluA2 expression. 131
Table 1. List of primers used for RT-qPCR in this study 132
Appendix 133
Appendix Figure 1. Repetitive intraocular injection affects the mRNA levels of innate immune genes in injected retinas. 134
Society of Neuroscience (SfN) 2018 Abstract and Poster 136
Institute of Molecular and Cellular Biology (IMCB) 2019 Abstract and Poster 139
dc.language.isoen
dc.title探討發育過程中麩胺酸訊息傳導對調控第二期視網膜波的影響zh_TW
dc.titleExploring the effects of glutamatergic transmission on regulating stage II retinal waves during developmenten
dc.typeThesis
dc.date.schoolyear107-2
dc.description.degree碩士
dc.contributor.oralexamcommittee徐立中(Li-Chung Hsu),盧主欽(Juu-Chin Lu),焦傳金(Chuan-Chin Chiao),陳示國(Shih-Kuo Chen)
dc.subject.keyword第二期視網膜波,麩胺酸,AMPA受體,GluA2,GluA2動態平衡,活體鈣離子顯像技術,zh_TW
dc.subject.keywordStage II retinal waves,Glutamate,AMPA receptor,GluA2,GluA2 dynamic,Live Ca2+ imaging,en
dc.relation.page141
dc.identifier.doi10.6342/NTU201902209
dc.rights.note有償授權
dc.date.accepted2019-08-05
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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