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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 | Pin-Chien Huang | en |
dc.contributor.author | 黃品蒨 | zh_TW |
dc.date.accessioned | 2021-06-15T06:46:37Z | - |
dc.date.available | 2016-07-06 | |
dc.date.copyright | 2011-07-06 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-06-16 | |
dc.identifier.citation | Adamic LA, Wilkinson D, Huberman BA, Adar E (2002) A literature based method for identifying gene-disease connections. Proc IEEE Comput Soc Bioinform Conf 1:109-117.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48117 | - |
dc.description.abstract | 在脊椎動物的視網膜發育過程中,存在一種自發性、有規律性的放電現象稱為「視網膜波」。腺苷酸是一種神經調節物質,在與細胞膜表面的接受器結合後,經由G蛋白質傳遞下游訊息;這些調節的作用,在整個視網膜波產生及傳遞的發育過程中都可以觀察到。之前的研究指出,腺苷酸接受器A2A亞型的促進劑及拮抗劑都會導致視網膜波的頻率增加。這種矛盾的結果,無法推論出腺苷酸接受器A2A亞型在視網膜波中所扮演的角色。另外,腺苷酸接受器A2A亞型是經由突觸前神經元(星狀無軸突細胞)或是經由突觸後神經元(視網膜節細胞)來調控視網膜波,也是待釐清的問題。因此,我們利用分子生物學的調控機制,探討在發育中大鼠的視網膜上腺苷酸接受器A2A亞型與視網膜波之間的關係。首先,我們確認在發育中的大鼠視網膜上內生的腺苷酸接受器A2A亞型表現在視網膜的內叢狀層及神經節細胞層,主要是在星狀無軸突細胞及視網膜節細胞的周圍。接著,利用整體的大量表現以及整體的降低表現,來調控腺苷酸接受器A2A亞型在視網膜上的表現量;藉由鈣離子影像技術去紀錄在腺苷酸接受器A2A亞型大量表現後,以及利用siRNA降低表現後的視網膜波現象。實驗結果發現,腺苷酸接受器A2A亞型整體的大量表現並不影響視網膜波的頻率;但是,整體降低腺苷酸接受器A2A亞型的表現會導致視網膜波的頻率下降。為了釐清腺苷酸接受器A2A亞型的調控因子,我們利用可以精確表現在星狀無軸突細胞的啟動子(mGluR2),調控腺苷酸接受器A2A亞型在星狀無軸突細胞的表現。藉由鈣離子影像技術以及全細胞膜片固定技術,我們發現在星狀無軸突細胞大量表現腺苷酸接受器A2A亞型會造成視網膜波的頻率增加,以及產生的間隔縮短。但是,其他的視網膜波特性,包含振幅、同步性以及時程都不受到腺苷酸接受器A2A亞型大量表現而影響。此外,視網膜節細胞的可興奮性、以及與視網膜波相關的突觸後細胞電流都不受到腺苷酸接受器A2A亞型大量表現而影響。因此,腺苷酸接受器A2A亞型的調控是經由突觸前神經元,而不會影響到突觸後神經元的性質。為了更進一步了解腺苷酸接受器A2A亞型調控的分子機制,我們利用兩種有突變缺陷的腺苷酸接受器A2A亞型大量表現在星狀無軸突細胞上;一種是讓腺苷酸接受器A2A亞型在與Gs蛋白質結合處遭受破壞,另一種是將腺苷酸接受器A2A亞型的C部尾端去除。實驗結果顯示,這兩種有缺陷的腺苷酸接受器A2A亞型突變,都無法造成跟大量表現原始的腺苷酸接受器A2A亞型相同的影響;也就是腺苷酸接受器A2A亞型在視網膜波上的調控,因為這兩處突變而沒有了作用。因此,藉由這些實驗結果,我們得知在發育時期,腺苷酸接受器A2A亞型是透過與Gs蛋白質結合以及C部尾端的交互作用,在突觸前神經元正向地調控視網膜波。 | zh_TW |
dc.description.abstract | Periodic and patterned spontaneous activity has been found in the developing vertebrate retina termed as “retinal waves”. Neuromodulator adenosine is involved in retinal wave generation and propagation throughout the entire developmental course and exerts its effects by activating its G-protein-coupled cell-surface receptors. In previous studies, both adenosine A2A receptor (A2AR) antagonist and agonist increased retinal wave frequency, yielding the results hard to interpret. Besides, it is unclear if adenosine A2AR modulates retinal waves via presynaptic (starburst amacrine cell, SAC) or postsynaptic (retinal ganglion cell, RGC) neurons. To address these questions, we studied the causal relationship between adenosine A2AR and retinal waves in the developing rat retina by molecular perturbation. First, we found endogenous adenosine A2AR is expressed in the inner plexiform layer and the ganglion cell layer (around SACs and RGCs) of the developing rat retina. Second, we changed the overall expression levels of adenosine A2AR in the retinal explant culture. Subsequent measurements of retinal waves were done by calcium imaging after overexpressing wild-type adenosine A2AR or knocking down its transcripts by the corresponding siRNA. We found that the overall overexpression of adenosine A2AR had no effect on wave properties, but the overall knockdown of adenosine A2AR decreased the retinal wave frequency, suggesting that endogenous adenosine A2AR positively modulates wave frequency. To verify if adenosine A2AR functions presynaptically, we applied a cell-type specific promoter, metabotropic glutamate receptor subtype II (mGluR2) promoter, to regulate adenosine A2AR expression in presynaptic neurons, SACs. By calcium imaging and whole-cell patch-clamp recordings, the retinal wave frequency was increased and the interval was decreased by overexpressing adenosine A2AR in SACs. In contrast, other wave properties, including amplitudes, correlations, and durations were not changed by overexpressing adenosine A2AR, suggesting the wave up-regulation by adenosine A2AR is via presynaptic modulation. To further delineate the underlying molecular mechanisms, we employed two dominant-negative mutants, the Gs-coupling defective mutant (A2AR-Gs*) and C-terminal truncated mutant (A2AR-ΔC). After overexpressing the mutant A2ARs in SACs, both dominant-negative adenosine A2ARs rescued the effects on retinal wave frequency and interval by the wild-type adenosine A2AR overexpression. Together, our results suggest that adenosine A2AR presynaptically up-modulates retinal waves via Gs-protein coupling and its C-terminal interaction. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T06:46:37Z (GMT). No. of bitstreams: 1 ntu-100-R98B43007-1.pdf: 5813031 bytes, checksum: c97f7436e3f575c4c005fd490ca7d2db (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | Contents
口試委員會審定書 i 誌謝 ii 中文摘要 iii Abstract v Abbreviations vii Contents x Chapter I Introduction 1 1.1 Neural Development 1 1.2 Organization of the Visual System 1 1.2.1 Anatomy of retina 2 1.2.2 Signal transduction of vision 2 1.3 Patterned Spontaneous Activity-Retinal Waves 3 1.3.1 Three distinct stages of retinal waves 4 1.3.2 Roles of retinal waves in visual system development 5 1.4 Adenosine Receptors 6 1.5 Adenosine A2A Receptors 6 1.5.1 Properties of adenosine A2A receptors 7 1.5.2 Distribution of adenosine A2A receptors 8 1.5.3 Signaling cascades underlying activation of adenosine A2AR 8 1.6 Adenosine Signaling in Retinal Waves 9 1.7 Techniques to Measure Retinal Waves 10 1.8 Objectives of This Study 11 Chapter II Materials and Methods 13 2.1 Vector Construction and Subcloning 13 2.2 Site-Directed Mutagenesis 15 2.3 Animals 16 2.4 Retinal Explant Culture 16 2.5 Transient Transfection-Electroporation 17 2.6 Calcium Imaging 17 2.7 Electrophysiology-Whole-cell Patch-Clamp 19 2.8 Immunofluorescence 20 2.9 Data Analysis 22 2.10 Statistics 23 Chapter III Results 24 3.1 Adenosine A2AR localized to IPL and GCL during stage II waves (P0-P9) 24 3.2 Overall overexpression of adenosine A2AR in the retina did not change retinal wave frequency 24 3.3 Overall knockdown of adenosine A2AR decreased retinal wave frequency 25 3.4 Expression of the mGluR2 promoter-driven constructs showed significant specificity in SACs as 69.4% compared to the CMV promoted as 10% 26 3.5 The interval of calcium waves was reduced by overexpression of wild-type adenosine A2AR in SACs 28 3.6 The interval of calcium waves was not changed by overexpressing adenosine A2AR dominant-negative mutants, i.e. the Gs-coupling defective mutant and the C-terminal truncated mutant 29 3.7 The properties of calcium waves were not changed by overall knockdown of adenosine A2AR, or overexpression of adenosine A2AR and its dominant-negative mutants in SACs 31 3.8 RGC membrane properties were not changed by adenosine A2AR 33 3.9 Other postsynaptic wave properties (durations, amplitudes, charges and the subthreshold depolarizations) were not changed by overexpression of adenosine A2AR and its dominant-negative mutants in SACs 33 Chapter IV Discussion 37 4.1 Endogenous adenosine A2A receptors in the developing rat retina 37 4.2 Presynaptic adenosine A2AR plays a positive role in regulating retinal waves 38 4.3 Presynaptic manipulation of adenosine A2AR does not alter the postsynaptic responsiveness in RGCs 40 4.4 Molecular mechanisms of adenosine A2AR in modulating retinal waves 42 Chapter V Conclusion 46 References 47 List of Figures Figure 1 The structure of the retina (Kolb, 2003) 52 Figure 2 Retinal waves (Firth et al., 2005) 53 Figure 3 Schematic representation of the human adenosine A2AR (modified from (Olah and Stiles, 2000)) 54 Figure 4 Experimental designs in this study 55 Figure 5 Data analysis of calcium waves 56 Figure 6 Pharmacological studies for adenosine A2AR modulation of retinal waves by whole-cell patch-clamp recordings (Wang CT, et al., unpublished data) 58 Figure 7 Retinal cross-sections with immunofluorescence labeling confirmed that adenosine A2AR localized to IPL during Stage II Waves (P0- P9) 59 Figure 8 Overall overexpression of adenosine A2AR in retina had no effect on retinal wave frequency 60 Figure 9 Knockdown of endogenous adenosine A2AR with siRNA reduced retinal wave frequency 62 Figure 10 Amplitudes and correlations of calcium waves were not changed by knockdown of adenosine A2AR 64 Figure 11 The levels of endogenous adenosine A2AR were decreased by siRNA knockdown 66 Figure 12 The mGluR2 promoter-driven constructs showed gene expression specific to SACs as 69.4% 67 Figure 13 Overexpression of adenosine A2AR in SACs increased retinal wave frequency, but the Gs-coupling defective mutant and C-terminal truncated mutant reversed the effect of wild-type adenosine A2AR 69 Figure 14 The properties of calcium waves were not changed by overexpression of wild-type or mutant adenosine A2AR 71 Figure 15 The levels of adenosine A2AR were increased after overexpression 74 Figure 16 The membrane properties of RGCs were not changed by overexpression of wild-type and mutant adenosine A2AR 75 Figure 17 Retinal waves in RGCs from whole-cell patch-clamp recordings 77 Figure 18 The properties of retinal wave recorded by whole-cell patch-clamp recordings 78 Figure 19 The working model 80 List of Tables Table 1 Homologous adenosine A2ARs identified from different species 81 Table 2 The list of primers 82 Table 3 The list of antibodies 83 Appendix The 2010 annual meeting of the Society for Neuroscience (San Diego, CA, U.S.A. 11/17-21/2010): poster and abstract 84 2011 Institute of Molecular and Cellular Biology Poster Contest: poster 86 | |
dc.language.iso | en | |
dc.title | 腺苷酸接受器A2A亞型調控發育中大鼠視網膜的自發性放電活動之分子機制 | zh_TW |
dc.title | Molecular Mechanisms underlying Adenosine Receptor A2A Subtype in Regulating Spontaneous Activity of the Developing Rat Retina | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳儀莊(Yijuang Chern),焦傳金(Chuan-Chin Chiao),連正章(Cheng-Chang Lien) | |
dc.subject.keyword | 視網膜波,自發性放電現象,腺苷,酸接受器A2A亞型,Gs蛋白質結合,神經發育,鈣離子影像技術,全細胞膜片固定技術, | zh_TW |
dc.subject.keyword | retinal waves,spontaneous activity,adenosine A2A recepto,Gs-protein coupling,neural development,calcium imaging,whole-cell patch-clamp, | en |
dc.relation.page | 86 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-06-21 | |
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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