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標題: | 發育大鼠的視網膜中第二期視網膜波與麩胺酸釋放的交互影響 Interaction between Stage II Retinal Waves and Glutamate Release in Developing Rat Retinas |
作者: | Ching-Yuan Yang 楊清媛 |
指導教授: | 王致恬 |
關鍵字: | 視網膜波,星狀無軸突細胞,視網膜節細胞,麩胺酸傳遞,Synaptotagmin I, retinal waves,starburst amacrine cells,retinal ganglion cells,glutamate transmission,Synaptotagmin I, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 視網膜波對於正確的視覺網路發育很重要,第二期視網膜波發生在新生大鼠第一天到第九天,其產生機制是由一群突觸前神經元-星狀無軸突細胞,週期性的釋放神經傳導物質例如:乙醯膽鹼和 γ-氨基丁酸至突觸後神經元-視網膜節細胞。先前的研究發現,突觸後視網膜節細胞可能分泌反向訊息到突觸前星狀無軸突細胞,進而影響視網膜波的頻率。而利用麩胺酸受體拮抗劑藥物,我們發現這個反向的調控訊號可能是視網膜節細胞釋放的麩胺酸。於是我們提出假設,麩胺酸可能從視網膜節細胞釋放,以逆行或自分泌的方式在視網膜內傳遞。為了確定麩胺酸在發育中視網膜的存在,我們使用免疫螢光染色以及細胞型態的麩胺酸光學感測器,發現麩胺酸存在於視網膜節細胞,並且大量釋放於視網膜內。為了確認視網膜波會調控麩胺酸的釋放,以及麩胺酸傳導的的方向,我們建立了麩胺酸的光學感測器技術,加以特異表達在視網膜節細胞或是星狀無軸突細胞。在視網膜節細胞或是星狀無軸突細胞中,麩胺酸的光學感測器的螢光強度皆會因為視網膜波的頻率增加而增強;代表第二期視網膜波會調控麩胺酸的釋放,並且麩胺酸的釋放會透過逆行以及自分泌的方式在視網膜內傳遞。接著,為了確認麩胺酸的傳遞對於第二期視網膜波的影響,我們在鈣離子顯像技術技術中使用不同的藥物。先前研究指出,一種腺苷酸受器促進劑會增加星狀無軸突細胞的胞吐作用,進而增加視網膜波的頻率。然而,此腺苷酸受器的促進劑所造成的視網膜波頻率的增加會被麩胺酸受體拮抗劑所抑制,說明了麩胺酸的傳導作用是星狀無軸突細胞胞吐作用的下游反應。此外,Synaptotagmin I被認為在鈣離子調控的胞吐作用中擔任鈣離子感應者的角色,先前研究指出將Synaptotagmin I鈣離子感應能力降低的突變株專一性大量表現在視網膜節細胞中,與控制組及Synaptotagmin I組別相比,會使視網膜波產生的頻率顯著下降。但是我們發現當周圍麩胺酸濃度為5 nM時,會使此突變株大量表現在視網膜節細胞中所造成的視網膜波頻率下降的現象消失,並且其視網膜波頻率增加後與控制組及Synaptotagmin I組別沒有顯著差異。綜合以上結果,我們提出在大鼠發育時期,第二期視網膜波與麩胺酸的釋放之間彼此存在著交互作用。第二期視網膜波的頻率被5 nM的麩胺酸所調控,且麩胺酸訊息會透過自分泌的方式被視網膜節細胞接收,或是逆行分泌的方式被星狀無軸突細胞接收,進而調控第二期視網膜波的時空特性。 Retinal waves are important for visual circuit development. During the first postnatal week in rats (P0-P9), stage II retinal waves are initiated by presynaptic starburst amacrine cells (SACs) releasing neurotransmitters to neighboring SACs and retinal ganglion cells (RGCs). Surprisingly, we previously found that postsynaptic RGCs may send a “retrograde” signal to presynaptic SACs, thus regulating wave frequency. Since the ionotropic glutamate receptor (iGluR) antagonists can significantly reduce the RGC-mediated increase in wave frequency, we thus hypothesized that glutamate signal from RGCs may function in a retrograde or autocrine manner. To determine the presence of glutamate in developing retina, we first performed immunofluorescence staining for the neurotransmitter glutamate and cell-based glutamate optical sensor. We found that glutamate was present in RGCs and volume released throughout the entire retina. To determine whether stage II retinal waves modulate glutamate transmission that may act in an autocrine or retrograde manner, we established the glutamate sensor specifically expressed in RGCs or SACs. The fluorescence intensity of both RGC-expressing and SAC-expressing glutamate sensors was significantly increased by enhancing wave frequency. The results suggest that the glutamate release is up-regulated by enhancing stage II waves, and the glutamate may transmit in an autocrine manner and a retrograde manner. To further determine the role of glutamate transmission in modulating stage II retinal waves, we applied various pharmacological reagents during Ca2+ imaging of retinal waves. The selective A2AR agonist (CGS) was previously found to increase the SAC exocytosis, thus increasing wave frequency. By contrast, the CGS-mediated increase in wave frequency was abolished by the iGluR antagonists, suggesting that glutamate transmission was downstream of the presynaptic effects on retinal waves. Moreover, Synaptotagmin I (Syt I), a Ca2+ sensor protein, can regulate retinal waves via Ca2+ binding to its Ca2+-binding domains. Overexpressing the weakened Ca2+ -binding mutant of Syt I, Syt I-D230S (Syt I-C2A*), in RGCs decreased wave frequency compared to Ctrl and Syt I. Bath-applying the ambient glutamate (5 nM) occludes the Syt I-C2A*-decreased effects on wave frequency via RGCs. Taken together, we conclude that the interaction exists between stage II retinal waves and RGCs-releasing glutamate in developing rat retinas. The frequency of stage II retinal waves can be modulated by 5 nM glutamate during the stage II period, and glutamate may serve as an autocrine signal to RGCs and a retrograde signal to SACs, further regulating spatiotemporal properties of stage II retinal waves. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7755 |
DOI: | 10.6342/NTU201702122 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 分子與細胞生物學研究所 |
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