2011-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/709021摘要：模式化自發性活動是發育中神經網路的獨特印記；在發育的視網膜上被稱為「視網膜波」。視網膜波出現於視覺開始前，具波動狀的時空性質，影響視網膜與中樞的連結；但引發的分子機制迄今不明。視網膜波由一群聯絡神經元(starburst amacrine cells, SACs)週期性的興奮所引起；SACs隨即釋放乙醯膽鹼至未成熟的節神經元(retinal ganglion cells, RGCs)，造成其週期性的動作電位、鈣離子濃度與PKA活性上升，並傳至RGC整層。然而，至今對於引起SACs內傳導物質釋放的機制尚待釐清。因傳導物質的釋放始於胞吐作用，故我們假設視網膜波的引發，與SACs內胞吐作用的分子機制有關，特別是synaptosomal-associated protein of 25 kDa (SNAP-25) 與 cysteine string protein (CSP)。支持的證據指出，在發育過程中，這些胞吐作用的相關分子可在不同的表現階段受到調控，包括轉錄啟始、轉錄後調控以及轉譯後修飾(磷酸化)。本計畫旨在闡明胞吐作用的相關分子與視網膜波之間的交互影響。藉由結合分子操作、單細胞反轉錄聚合&#37238;連鎖反應、初級視網膜離體培養、全細胞膜電位測量及活細胞即時影像，回答以下問題：SACs內SNAP-25或CSP的表現量與磷酸化狀態能否調控視網膜波的模式？改變視網膜波的模式能否改變SACs內SNAP-25或CSP的表現量？由何訊息路徑？這些分子的表現與視網膜波的起落有何時間上的相關性？藉由解答這些問題，我們可瞭解視網膜波是如何被引發的，進而認識神經網路發育的普遍機制。<br> Abstract: During the critical period of neural circuit refinement, the recurrent, patterned and spontaneous activity is a unique hallmark in many parts of the developing nervous system. This patterned spontaneous activity has been studied vigorously in the developing retina termed as retinal waves. Retinal waves occur prior to visual experience with wave-like spatiotemporal patterns. Although retinal waves have been shown essential for establishing functional connections between the retina and its central brain targets, the molecular mechanisms underlying wave initiation remain largely unknown. Extensive studies have suggested retinal waves start with periodic firings of a subset of retinal interneurons (starburst amacrine cells, SACs) during the first week of postnatal rodents. SACs then release acetylcholine (ACh) onto immature retinal output neurons (retinal ganglion cells, RGCs) to cause rhythmic bursts of action potentials, calcium transients and PKA activities propagating across the entire RGC layer. However, it remains elusive at the cellular level how the periodic firings of action potentials can modulate neurotransmitter release from SACs. Since neurotransmitter release is mediated by calcium-regulated exocytosis, we hypothesize that the initiation of retinal waves is associated with the changes in the exocytotic machinery in SACs, in particular, synaptosomal-associated protein of 25 kDa (SNAP-25) and cysteine string protein (CSP). Supporting evidences indicate that during development these exocytosis-related molecules can be regulated at different expression stages, including transcriptional activation, post-transcriptional control and post-translational modification (phosphorylation). Therefore, the goal of this proposal is to elucidate how these exocytosis-related molecules affect retinal wave activity, and vice versa. By combining molecular perturbation, single-cell reverse-transcriptase quantitative PCR (sc RT-qPCR), primary retinal explant culture, whole-cell patch clamp and live imaging, we will address the following questions. First, are retinal waves regulated by the changes in the expression levels or phosphorylation states of SNAP-25 and CSP in SACs? Second, can the altered patterns of retinal waves change the expression levels of these exocytosis-related molecules in SACs? Via which underlying signaling pathway(s)? Finally, how is the expression of these molecules temporally associated with the up/down-phase of retinal waves? By addressing these questions, we can understand how retinal waves occur during retinal development. The results from this research proposal will provide the advanced understanding for the general mechanism in neural circuit development.神經網路發育模式化自發性活動視網膜波Starburst amacrine cells (SACs)Synaptosomal-associated protein of 25 kDa (SNAPNeural circuit developmentPatterned spontaneous activityStarburst amacrine cells (SACs)Synaptosomal-associated protein of 25 kDa (SNAP-25)Cysteine string protein (CSP)