摘要:神經網路發育時,會出現模式化的自發性放電現象,使網路正確連結。此現象在發育的視網膜被稱為「視網膜波」;以藥物抑制視網膜波會使視神經與中樞的連結錯誤。視網膜波由一群特定的神經元(Starburst Amacrine Cells, SACs)週期性釋放傳導物質至節神經元(Retinal Ganglion Cells, RGCs)所造成,使得RGCs的動作電位與鈣離子濃度週期性上升;雖然視網膜波具有波動狀的時空模式,但其模式的調控機制並不清楚。研究顯示以藥物降低PKA的活性會縮小視網膜波的規模;但詳細機制迄今不明。若PKA會藉由磷酸化蛋白以調控視網膜波的模式,其中最有可能的蛋白應涉及SACs內傳導物質的釋放;目前文獻上僅有記載Cysteine String Protein (CSP) 與Synaptosome-Associated Protein of 25 kDa (SNAP-25)為PKA Substrates。本計畫旨在探討PKA的磷酸化蛋白如何調控視網膜波的模式。藉由結合分生技術、初級培養、電生理、即時影像、單細胞反轉錄聚合酶連鎖反應,回答以下問題: (一) 何種特定的視網膜波模式可能被SACs中的被PKA磷酸化的CSP或SNAP-25所調控? (二) 磷酸化的CSP或SNAP-25透過何種機制以影響特定的視網膜波模式? (三) 是否還有其他的分子會被PKA磷酸化而改變視網膜波的模式? 是影響突觸前的SACs,或是突觸後的RGCs?藉由解答這些問題,我們可解析視網膜波模式調控的機制;以利未來研究不同模式如何影響視神經網路的連結,進而瞭解網路發育的普遍機制。
Abstract: During a developmental critical period, the developing neural circuits display a unique hallmark, i.e., patterned spontaneous activity, to ensure precise connections of neural circuits. This patterned spontaneous activity has been studied vigorously in the developing retina termed “retinal waves”. Blocking retinal waves prior to visual experience leads to the aberrant retinal projections to central targets. Retinal waves are initiated by a subset of retinal neurons (starburst amacrine cells, SACs) periodically releasing neurotransmitters onto retinal ganglion cells (RGCs), thus rhythmic action potentials and calcium transients propagating across the entire RGC layer. Although retinal waves confer the wave-like spatiotemporal patterns, the mechanisms regulating wave patterns remain elusive. Preliminary results suggest that a decrease in the PKA activity reduced the frequency, size, and propagation speed of retinal waves. However, the mechanisms underlying PKA modulation of wave patterns are completely unknown. Here, we hypothesize that PKA modulation of wave patterns is mediated through phosphorylation of PKA substrates, probably the proteins mediating neurotransmitter release in SACs. To date, among the proteins involving neurotransmitter release, only cysteine string protein (CSP) and synaptosome-associated protein of 25 kDa (SNAP-25) have been shown as PKA substrates. Therefore, the goal of this proposal is to elucidate how the PKA substrates can modulate the patterns of retinal waves upon phosphorylation. By combining molecular biology, primary retinal explant culture, whole-cell patch clamp, live imaging, and single-cell reverse transcriptase-PCR (sc RT-PCR), we will address the following questions. First, which spatial or temporal patterns of retinal waves can be regulated by changing the phosphorylation states of CSP and SNAP-25 in SACs? Second, by which mechanisms can the phosphorylated CSP and SNAP-25 regulate the patterns of retinal waves? Third, are there any other PKA substrates in the developing retina involving modulation of retinal wave patterns? Do these PKA substrates act via presynaptic SACs to affect the releasing mechanism? Or via postsynaptic RGCs to affect the responsive mechanism? By addressing these questions, we will delineate the mechanisms underlying modulation of retinal wave patterns. The results from this proposal will provide the foundation to further explore how wave patterns may affect visual circuit formation, and thus we can understand the general rules for neural circuit refinement.