Synaptotagmin I在發育中大鼠視網膜的神經節細胞內調控模式化自發性的放電現象

Synaptotagmin I in RGCs regulates the patterned spontaneous activity in the developing rat retina

楊政璋(Cheng-Chang Yang)

指導教授 ： 王致恬

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.11022

摘要

在脊椎動物發育中的視覺系統內，存在著一種模式化、規律性的放電現象被稱為「視網膜波」，它主要發生於視覺開始之前並且具有獨特的波動狀時空性質，使得正確的視覺網絡能夠形成。目前已經知道視網膜波的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，造成其膜電位、鈣離子濃度及下游PKA活性週期性的振盪，最終導致基因表現，造成雙眼連結網路的分離以及視野的區隔。倘若以藥物抑制了視網膜波的產生，則會使得視神經與大腦中樞的連結嚴重受損，因此，了解調控視網膜的產生將有助於研究視覺發育。
星狀無軸突細胞主要藉由鈣離子調控的胞吐作用釋放神經傳導物質，而參與該作用的蛋白質包含了SNARE蛋白以及Synaptotagmin (Syt)。目前在Syt家族裡面有17種亞型蛋白質，其中的Syt 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，而先前的研究已經證實Syt I在星狀無軸突細胞中利用其上的兩個C2鈣離子結合區域－C2A及C2B－調控視網膜波的時空特性，另一方面，在視網膜節細胞及視神經中也分別發現了Syt I的轉錄產物及蛋白質的存在，然而，是否視網膜節細胞中的Syt I也能利用鈣離子調控的胞吐作用影響視網膜波的時空性質仍需更進一步的研究。
在本研究中，我們利用大鼠出生一周內的視網膜作為研究材料，在這期間是視網膜波造成視覺發育的關鍵時期。藉由免疫螢光染色發現Syt I表現在內網狀層及視網膜節細胞層中，而在單顆的視網膜節細胞內更發現它位於突觸囊泡及緻密核心囊泡上，這說明了Syt I位於視網膜節細胞中可能可以調控神經傳導物質的釋放。為了瞭解Syt I在視網節細胞中所扮演的角色，我們使Syt I及其鈣離子感應能力降低的突變株(Syt I-C2A*及Syt I-C2B*)專一性的表現在視網膜節細胞中，再利用鈣離子顯像技術紀錄發生於視網膜節細胞層的視網膜波產生的鈣離子變化。結果發現，與控制組相比，當大量表現Syt I時，視網膜波產生的頻率顯著增加，而只有大量表現Syt I-C2A*時才會使得視網膜波產生的頻率顯著下降。此外，就視網膜波在細胞之間傳遞的同步性而言，與Syt I-C2A*相比，大量表現Syt I時大幅降低了同步性質。因此這些結果說明了視網膜節細胞內的Syt I具有調控視網膜波的時空性質的功能，意謂著其可能具有調控分泌反向訊息的功能，進而影響星狀無軸突細胞改變釋放神經傳導物質的機制以及視網膜波的時空模式，而利用藥物實驗，我們發現這個反向的調控訊號是視網膜節細胞釋放的麩胺酸。綜合以上的結果，我們的研究首度指出視網膜節細胞不僅身為視網膜中的唯一的輸出神經元，也同時扮演著藉由其內的Syt I釋放麩胺酸來調控視網膜波產生的角色。

關鍵字

Synaptotagmin I ； 視網膜波 ； 星狀無軸突細胞 ； 視網膜節細胞 ； 麩胺酸 ； 鈣離子顯像技術

並列摘要

During a critical period of the developing vertebrate visual system, patterned spontaneous activity, i.e., retinal waves, is required to sculpture and refine vertebrate visual circuits prior to the onset of vision. Retinal waves confer the wave-like spatiotemporal patterns and are mediated by cholinergic neurotransmission during the first week of postnatal development in rodent. At this stage, cholinergic interneurons, presynaptic starburst amacrine cells (SACs), undergo Ca2+-regulated exocytosis to release excitatory neurotransmitters, acetylcholine (ACh) and γ-amino butyric acid (GABA), onto neighboring SACs and postsynaptic retinal ganglion cells (RGCs). A previous study showed that in the developing SACs, a Ca2+ sensor protein, Synaptotagmin I (Syt I), can regulate the temporal patterns of retinal waves via Ca2+ binding to its Ca2+-binding domains, C2A and C2B. However, Syt I’s expression is also found in postnatal RGCs and optic nerves (mainly composed of RGC axons). Thus, whether Syt I in RGCs is involved in regulating retinal waves remains unknown. To address this question, we explored the functional role of Syt I in RGCs from postnatal P0 to P9 in rats, by combining molecular perturbation, immunofluorescence staining, live Ca2+ imaging, and pharmacological manipulation.
We found that Syt I was expressed in both inner plexiform layer (IPL) and GCL of the developing rat retina during stage-II retinal waves. The expression of Syt I in dissociated RGCs localized to secretory vesicles, implying that Syt I may regulate the secretion of neurotransmitter in RGCs. To further study the relationship between Syt I in RGCs and retinal waves, we manipulated Syt I molecules by overexpressing Syt I and its weakened Ca2+-binding mutants, Syt I-D230S (Syt I-C2A*) and Syt I-D363N (Syt I-C2B*), in RGCs. We used ex vivo transfection with the Brn3b promoter-driven gene expression to target molecular perturbation exclusively to RGCs. By measuring Ca2+ transients, we found that overexpression of Syt I in RGCs altered spatiotemporal properties of retinal waves, including increasing wave frequency, reducing wave size, and decreasing spatial correlation. By contrast, overexpression of both Syt I-C2A* and Syt I-C2B* in RGCs decreased wave frequency compared to Syt I, but only Syt I-C2A* had significant reduction compared to Ctrl. Moreover, wave spatial correlation was significantly different between Syt I and Syt I-C2A* in the cell pairs located at near positions. Based on these results, we suggest that Ca2+ binding to the C2-domains of Syt I in RGCs, mainly C2A domain, may provide a new form of retrograde plasticity during the development of neural circuits. Finally, pharmacological experiments revealed that Syt I’s up-regulation of wave frequency via RGCs is mediated by glutamate secreted from RGCs. Thus, we conclude that during cholinergic waves, glutamate is secreted from RGCs through Syt I’s action, mainly via Ca2+ binding to the C2A domain, thus increasing the excitability of SACs and enhancing wave frequency. Our results provide the evidence contrary to the conventional idea that RGCs only send the “output” signals from retinas to central brain.

並列關鍵字

Synaptotagmin I ； retinal waves ； starburst amacrine cells ； retinal ganglion cells ； glutamate ； live Ca2+ imaging

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