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標題: | 第三型突觸連結蛋白的鈣離子結合能力調控模式化自發性放電現象和視覺網路發育 Calcium binding to Synaptotagmin III regulates patterned spontaneous activity and visual circuit development |
作者: | Wen-Chi Shu 舒玟綺 |
指導教授: | 王致恬 |
關鍵字: | 突觸連結蛋白三,第二期視網膜波,麩胺酸釋放,雙眼隔離現,鈣離子即時影像技術,活體電穿孔技術, Synaptotagmin III,Stage II retinal waves,Glutamate release,Eye-specific segregation,Calcium imaging,In vivo electroporation, |
出版年 : | 2016 |
學位: | 碩士 |
摘要: | 視覺網路發育的過程中,視網膜上的神經細胞會出現大規模、有規律性的自發性放電現象,稱為視網膜波。過去的研究顯示,第二期視網膜波對於完成視網膜至腦部的投射非常重要,這個時期的視網膜波由星狀無軸突細胞(Starburst amacrine cells; SACs)釋放乙醯膽鹼及-氨基丁酸(GABA)至視網膜節細胞(Retinal ganglion cells; RGCs),RGCs會進而產生神經衝動並傳遞至腦部不同的投射區,其中研究較透徹的為背外側膝狀體(dorsal-lateral geniculate nucleus; dLGN),會有特殊的兩眼投射顯著分離現象(eye-specific segregation)。以大鼠來說,第二期視網膜波發生於仔鼠出生當天至出生後第九天,其中最重要的時期約在出生後第四至第八天,在此時期中沒有神經傳遞或者神經傳遞訊號較弱的神經末梢會被修剪,使訊號能夠集中投射且與腦部的神經元有較緊密的結合。目前普遍認為視網膜的時空傳播特性是完成正確投射的線索,例如視網膜波的頻率、多少細胞一起參與或一次的自發性放電有多少電流的湧入……等。雖然我們尚不了解視網膜波與正確投射的形成是由那些傳播特性所調控,但之前發現只要此時期的波有部分特性的改變,就容易改變視神經投射區的分離現象。
在我們實驗室過去的研究中發現第三型突觸連結蛋白(Synaptotagmin III; Syt III),會於仔鼠出生後第四至六天大量表現於RGCs,除此之外,Syt III也會表現於SACs中。Syt III是屬於Syt家族中的一員,它們大多被發現在神經細胞中,並扮演著接收鈣離子引發胞吐作用的角色。因為Syt III與釋放神經傳導物質相關,在此篇研究中我們利用過量表現Syt III或其突變株以及紀錄視網膜波同步的鈣離子動態變化,探討Syt III在視網膜波中所可能扮演的角色。首先,我們將正常的Syt III與降低鈣離子接收能力的Syt III 突變株(Syt III-C2AB*)過量表現於SACs或RGCs中,觀察其視網膜波是否有時空特性的改變。結果顯示,不管是在SACs或是RGCs中,過量表現Syt III-C2AB*與Syt III的組別相比,其視網膜波的頻率會下降;除此之外,RGCs中,Syt III也會降低鄰近細胞一起參與視網膜波的同步性 (亦即降低空間傳遞範圍)。根據此研究我們得知,Syt III會透過與鈣離子的結合調控視網膜波的傳遞。 因為Syt III會在RGCs中影響模式化放電現象的空間傳播特性,我們藉由文獻與相關研究推論Syt III在RGCs中可能藉由釋放麩氨酸(glutamate)影響SACs,進而調控視網膜波的傳播。因此,我們在視網膜波的紀錄中加入離子型麩氨酸受器拮抗劑(ionotropic glutamate receptor antagonists, iGluR antagonists),發現Syt III過量表現於RGCs中所造成視網膜波頻率增加的效應會被抑制,因而得知Syt III在RGCs中能藉由glutamate的釋放增加視網膜波的頻率。最後,為了探討Syt III在RGCs中,若表現於視網膜發育的關鍵時期對視神經投射的影響,我們設計了在活體內表現外源性DNA的方式,配合追蹤染劑的使用,發現Syt III若表現於單一眼,同側dLGN的分離現象會顯著下降。因此,我們發現Syt III表現於RGCs可調控第二期視網膜波的傳遞,並進而影響同側腦區的投射。 Patterned spontaneous activity in developing retina, termed retinal waves, appears during the critical period of visual circuit refinement. Stage II retinal waves, critical for establishing the eye-specific segregation of retinogeniculate and retinocollicular projections, are mediated by neurotransmitter release from starburst amacrine cells (SACs) via Ca2+-dependent exocytosis, affecting neighboring SACs and retinal ganglion cells (RGCs). We previously found that the expression of synaptotagmin III (Syt III), a Ca2+ sensor protein in vesicle release, is upregulated in rat RGCs and optic nerves during P4-P6 (P4-P8 in rodents as the critical period for eye-specific segregation). Moreover, Syt III regulates the kinetics of Ca2+-dependent exocytosis through Ca2+ binding to the C2A and C2B domains. However, how and why Ca2+ binding to Syt III regulates stage II waves remain unknown. In this study, we overexpressed Syt III or Syt III-C2AB* (a mutant harboring the abolished Ca2+-binding sites) in SACs or RGCs by the cell-type specific promoters, and further performed live Ca2+ imaging to measure the subsequent changes on wave properties. First, we found that overexpressing Syt III-C2AB* in SACs decreased the wave frequency compared to Syt III. Moreover, in neonatal RGCs, Syt III dramatically increased the frequency and decreased the spatial correlation of retinal waves, but Syt III-C2AB* did not. These results suggest that Syt III in SACs or RGCs can regulate the kinetics of retinal waves through Ca2+ binding to its C2AB domains. However, Syt III in RGCs plays a relatively profound role in wave regulation compared to Syt III in SACs. Previous studies showed that RGCs are the source of glutamate release in developing retinas. To further investigate how Syt III in RGCs modulates stage-II waves, we applied ionotropic glutamate receptor antagonists and found that the frequency of wave-associated Ca2+ transients was decreased to the same level in retinas overexpressing control, Syt III, or Syt III-C2AB*. Therefore, Syt III in developing RGCs can promote glutamatergic transmission between RGCs and SACs, thus affecting the wave properties. Finally, by using in vivo electroporation, we transfected P3 rat RGCs with Syt III and found the aberrant pattern in ipsilateral retinogeniculate projections. Together, our results suggest that Syt III in developing RGCs plays an important role in regulating patterned spontaneous activity and visual circuit development during a critical development period. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50578 |
DOI: | 10.6342/NTU201601240 |
全文授權: | 有償授權 |
顯示於系所單位: | 分子與細胞生物學研究所 |
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