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標題: | Synaptotagmin III在大鼠視網膜的發育過程中所扮演的角色 The Functional Role of Synaptotagmin III in the Developing Rat Retina |
作者: | Shao-Yen Kao 高韶巖 |
指導教授: | 王致恬 |
關鍵字: | Synaptotagmin III,視網膜波,規律自發性放電反應,視野分離,鈣離子影像技術, synaptotagmin III,retinal waves,patterned spontaneous activity,eye-specific segregation,calcium imaging, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 雙眼視覺神經發育過程中,視覺神經網路修飾需要在視網膜引發的一系列規律性、自發性的放電反應,稱之為「視網膜波」。視網膜波在發育的關鍵期(大鼠為出生後四天到八天)會影響到視網膜節細胞的軸突投射到腦區,而形成雙眼視野分離的現象。在大鼠出生剛出生到出生後的第九天,視網膜波的調控是由一群突觸前細胞(星狀無軸突細胞)釋放出乙醯膽鹼到突觸後的視網膜節細胞,此時期稱為第二期視網膜波。因此,改變星狀無軸突細胞的釋放機制可以調控第二期視網膜波。 Synaptotagmin (Syt) 家族是一種鈣離子感應蛋白,可以引發鈣離子調節的胞吐作用來影響神經傳導物質的釋放。目前為止Syt家族總共發現有17種不同的isoforms。先前的研究得知,Syt I在第二期視網膜波時期,可透過其 C2AB Domain 和鈣離子結合,正向地調控視網膜波。然而,在第二期視網膜波中,其他 Syt isoforms在視網膜內表現的情形和功能至今並不清楚。在本研究中,我們偵測 Syt isoforms (Syt I, III, IV) 在發育的視網膜以及視神經中的時空表現模式,以進而探討其在視網膜發育過程中可能的功能。研究結果發現,在視網膜發育過程中, Syt I 在視網膜內側以及視神經中的表現相對穩定,而 Syt III 在形成視野分離的關鍵時期中,於視網膜內側以及視神經中的表現模式和表現量會隨著發育過程有顯著性的變化。因此,我們專一研究 Syt III 是否會調控視網膜波;利用分子干擾技術以及活細胞鈣離子顯像技術,我們可以瞭解 Syt III 在視網膜波中所扮演的角色。藉由去測量與視網膜波相關的鈣離子流的性質,我們發現,在突觸前星狀無軸突細胞或突觸後視網膜節細胞內大量表現 Syt III都會讓視網膜波的鈣離子流的頻率顯著增加。此外,在突觸後視網膜節細胞內大量表現 Syt III 會減弱鈣離子流在細胞之間傳遞的同步性;相反的,減少Syt III 在視網膜節細胞的表現可以增加空間傳遞的同步性質。因此,我們的研究結果顯示,在發育大鼠的視網膜中,Syt III可分別在突觸前星狀無軸突細胞以及突觸後視網膜節細胞,不同地調控第二期視網膜波的時間與空間性質;因此,Syt III可能在視野分離中扮演重要的角色。 Binocular visual circuit refinement requires a robust patterned spontaneous activity in the developing retina, termed as retinal waves. During the developmental critical period (P4 - P8 in rodent), retinal waves modulate the axon projection of retinal output neurons (retinal ganglion cells, RGCs) to their central brain targets, so visual circuits can be refined as eye-specific segregation. In postnatal rodent (P0 - P9), retinal waves are initiated via a subset of interneurons (starburst amacrine cells, SACs) releasing acetylcholine (ACh) onto postsynaptic RGCs (so-called stage-II waves). Therefore, altering the releasing mechanism in presynaptic SACs can modulate stage-II waves. Synaptotagmin (Syt) family serves as a Ca2+ sensor during Ca2+-regulated exocytosis to mediate neurotransmitter release. To date, more than seventeen Syt isoforms have been discovered. Our previous study has shown that Syt I positively regulates stage-II waves by Ca2+ binding to its C2AB domains. However, the expression and function of other Syt isoforms remain elusive during stage-II waves. In the study, we investigated the spatiotemporal expression pattern of Syt isoforms (I, III and IV) in the developing rat retina and optic nerve, in order to explore their possible functions during development. We found that distinct from Syt I, Syt III was expressed in a unique pattern in inner retinas and optic nerves, and the expression levels were dramatically increased during the period of eye-specific segregation. Thus, we focused on the role of Syt III in regulating retinal waves. Whole-mount rat retinas (P0 - P2) were transfected with the DNA vectors carrying Syt III or antisense-Syt III in presynaptic SACs or RGCs. Control was the vector carrying the transfection marker EGFP. By measuring the Ca2+ transients associated with retinal waves, we found that the Ca2+ transient frequency was significantly increased by Syt III overexpression in either SACs or RGCs compared to control. Moreover, the spatial correlation of retinal waves was dampened by Syt III overexpression in RGCs but not in SACs, whereas Syt III knockdown in RGCs increased the spatial correlation. Thus, our results suggest that Syt III may differentially regulate the temporal and spatial properties of stage-II waves via SACs or RGCs in the developing rat retina. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/62161 |
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顯示於系所單位: | 分子與細胞生物學研究所 |
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