摘要:神經網路發育時會展現獨特的模式化自發性活動;在發育中的視覺網路稱為視網膜波,其功用為連結視網膜至中樞的神經網路。視網膜波出現於視覺開始前的關鍵期,此時的視網膜中多為神經母細胞 (neuroblasts),僅含有兩種已分化完全的神經元‒亦即星狀無軸突細胞 (starburst amacrine cells, SACs) 和視網膜神經節細胞 (retinal ganglion cells, RGCs)。已知視網膜波由突觸前神經元SACs釋放乙醯膽鹼至突觸後RGCs所引發,並以波動狀的時空特性傳播至整個視覺網路。本實驗室之前的研究證實SACs內的胞吐作用分子可調控視網膜波的傳播模式,例如:引介胞吐作用的鈣離子感應蛋白Synaptotagmin (Syt) 的無功能突變株至SACs內,可降低視網膜波的發生頻率;顯示視網膜波的發生與SACs中的胞吐作用機制有關。然而,迄今未知的是,突觸後RGCs如何影響視網膜波的時空特性?初步結果顯示在視網膜波出現的關鍵期內,不同Syt isoforms在RGCs的表現量會被精準地調控;且改變RGCs內的Syt isoforms可調控視網膜波的時空特性,此效應會進一步被麩胺酸接受器的抑制劑所廢除。因此,我們假設RGCs可釋放麩胺酸至視網膜中,調控視網膜波的特性並進而影響視覺網路的發育。本研究藉由結合初級視網膜離體培養、分子操作、單細胞反轉錄聚合酶連鎖反應、全細胞電位測量、活細胞即時影像、活體電穿孔及軸突追蹤技術,探討以下問題:RGCs是否釋放麩胺酸至視網膜中並進而調控視網膜波的時空特性?RGCs釋放的麩胺酸是藉由反向信號的作用以調控SACs的釋放功能進而影響視網膜波?抑或是藉由調控RGCs本身細胞膜的興奮性以影響視網膜波?調控RGCs的釋放機制是否可進一步影響視覺網路的發育?藉由解答這些問題,我們可瞭解視網膜節細胞在調控視覺網路發育上所扮演的角色;因傳統上認為RGCs僅是視網膜的信號輸出細胞,此研究成果將突破既有的觀念,揭開關於視覺網路發育新的分子機制。
Abstract: Patterned spontaneous activity is the unique hallmark present in the developing vertebrate neural circuits. The best characterized patterned spontaneous activity is “retinal waves”, which are essential for establishing precise retinal projections to brain targets. During the developmental critical period prior to visual experience, retinas consist of neuroblasts and two types of differentiated neurons, i.e., starburst amacrine cells (SACs) and retinal ganglion cells (RGCs). Retinal waves are initiated by acetylcholine release from presynaptic SACs onto postsynaptic RGCs, propagating through the entire visual system with distinct spatiotemporal properties. We previously reported that molecular manipulations in the developing SACs lead to the changes in the patterns of retinal waves in the postnatal rat retina. Particularly, introducing the dominant-negative mutants of synaptotagmin I (Syt I) that diminishes Ca2+-dependent exocytosis in SACs can further dampen the frequency of retinal waves, indicating that patterned regulation of retinal waves is associated with exocytotic mechanisms in presynaptic SACs. However, how postsynaptic RGCs can modulate retinal waves remains unknown.
Our preliminary data show that the expression of certain Syt isoforms is precisely regulated in developing RGCs during the wave period. Moreover, modulation of the releasing machinery in developing RGCs further affects the spatiotemporal properties of retinal waves, but these effects are abolished by bath-applying the ionotropic glutamate receptor antagonists. Here, we hypothesize that glutamate released from RGCs can modulate the patterns of retinal waves and further affect visual circuit development. In this proposal, we will identify the role of glutamate released by RGCs in regulating retinal waves and visual circuit development. By combining primary retinal explant culture, molecular perturbation, single-cell quantitative reverse transcriptase-PCR (sc RT-qPCR), whole-cell patch clamp, live imaging, in vivo electroporation, and axonal tracing, we will study the following questions. Is glutamate directly released from RGCs to modulate retinal waves? What are the cellular mechanisms by which the RGC-releasing glutamate can modulate the patterns of retinal waves? Through acting as a retrograde signal to regulate the secretion of SACs? Or through modulating the membrane excitability of RGCs? How can the RGC-releasing glutamate within retinas affect visual circuit development? Through addressing these questions, we will understand how RGCs regulate visual circuit development. Since RGCs are typically considered as retinal “output” neurons, but rather sending the retrograde signal back toward retinas. The results from this research proposal will provide the conceptual advance for the novel mechanism underlying visual circuit development.