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  1. NTU Theses and Dissertations Repository
  2. 生命科學院
  3. 分子與細胞生物學研究所
Please use this identifier to cite or link to this item: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72457
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
Exploring the effects of glutamatergic transmission on regulating stage II retinal waves during development
Authors: Sheng-Ping Hsu
徐聖平
Advisor: 王致恬(Chih-Tien Wang)
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,
Stage II retinal waves,Glutamate,AMPA receptor,GluA2,GluA2 dynamic,Live Ca2+ imaging,
Publication Year : 2019
Degree: 碩士
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。
During 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.
URI: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72457
DOI: 10.6342/NTU201902209
Fulltext Rights: 有償授權
Appears in Collections:分子與細胞生物學研究所

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