第三型突觸連結蛋白調控模式化自發性放電現象與視覺網路發育的機制

Abstract

在發育初期的雙眼視覺系統中，來自兩眼的視覺訊號會交疊在大腦中兩側的背外側膝狀體(dLGN, dorsal lateral geniculate nucleus)。隨著視覺網路的發育，在dLGN交疊的雙眼視覺訊號會漸漸分開來，而最終雙眼視覺訊號在dLGN會各自佔據一塊互不交疊的區域，而此現象稱作兩眼投射分離現象(eye-specific segregation)。數十年來，研究指出兩眼投射分離現象主要由第二期發育視網膜中的自發性放電現象所調控，而此放電現象簡稱為視網膜波。第二期視網膜波是由星狀無軸突細胞(SACs, starburst amacrine cells)釋放乙醯膽鹼(ACh, acetylcholine)刺激周邊的SACs與視網膜節細胞(RGCs, retinal ganglion cells)，而RGCs進而產生神經衝動並將視網膜中的訊息傳遞至中樞神經系統中不同腦區，如本篇研究的dLGN。在本篇研究當中，我們探討Synaptotagmin III第三型鈣離子突觸結合蛋白(Syt3)在發育中視網膜如何調控第二期視網膜波以及下游腦區的視覺迴路。Syt主要存在於突觸前及突觸後神經元，藉其鈣離子結合位點與鈣離子結合，以調控在突觸前神經元神經傳導物質的釋放。而目前已在中樞神經中發現17種Syt，而每種Syt在調控突觸間物質的釋放上有各自的差異性。Syt3在過去實驗室研究中發現，在出生後大鼠的RGC中，Syt3在第二期視網膜中短暫地大量表現於調控兩眼投射分離現象的關鍵時期，因此我們假設RGC中的Syt3在調控第二期視網膜波及下游的視覺迴路上扮演一重要角色。因此我們利用點突變、體外電穿孔轉染、活體電穿孔轉染、活細胞鈣離子顯像技術、冷凍切片、螢光染色等技術來證明此假設。 首先，我們利用體外電穿孔轉染在RGC中大量表現正常的Syt3及兩個鈣離子結合蛋白各自突變的Syt3 變異株(Syt3-C2A*及Syt3-C2B*)，我們發現正常的Syt3與其變異株在調控第二期視網膜波的時空特性上，有相似的結果。其二，我們利用活體電穿孔轉染在RGC中大量表現正常的Syt3以及會轉錄出反序列且互補的Syt3片段以干擾及降低原生Syt3的表現。我們發現正常的Syt3在調控第二期視網膜波的時空特性上並沒有顯著的差異；反之，降低原生Syt3的表現會使第二期視網膜波中鈣離子流入的持續時間與強度下降。其三，我們接著探討由Syt3影響的第二期視網膜波如何影響下游的視覺迴路形成。結果顯示，在RGC中降低原生Syt3的表現會改變大腦兩側dLGN中的兩眼投射分離現象。其四，從過去實驗數據推論麩胺酸在調控第二期視網膜波可能扮演重要角色。因此我們間隔性施打離子型麩胺酸受體拮抗劑(iGluR Antagonists)以影響第二期視網膜波及觀察dLGN中的兩眼投射分離現象。結果發現在第二期視網膜波中，受影響的麩胺酸訊息傳遞會改變在大腦同側dLGN中的兩眼投射分離現象。從上述結果，我們推論Syt3在調控第二期視網膜波及兩眼投射分離現象上扮演重要角色；且在視網膜中，正常的麩胺酸訊息傳遞對兩眼投射分離現象也相當重要。

In the developing binocular visual system, axon terminals projecting from two eyes are initially evenly innervated in a region of thalamus, i.e., the dorsal lateral geniculate nucleus (dLGN). During a developmental critical period (P4-P8 in rodent), the innervation of unwanted terminals in the dLGN is further refined, leading to the eye-specific segregation of the dLGN. Previous studies found that the eye-specific segregation of the dLGN requires patterned spontaneous activity in the developing retina, termed stage II retinal waves, propagating through developing starburst amacrine cells (SACs) and retinal ganglion cells (RGCs) to the visual cortex. We previously found synaptotagmin III (Syt3), a Ca2+ sensor, is transiently overexpressed in RGCs during the developmental critical period of eye-specific segregation and stage II retinal waves. However, how Syt3 in developing RGCs regulates stage II retinal waves and the eye-specific segregation of the dLGN remains unclear. To study the role of the Ca2+-binding domains (C2A and C2B) of Syt3 in regulating stage II retinal waves, we overexpressed Syt3-C2A* or Syt3-C2B* in RGCs and found that both mutants did not significantly alter stage II retinal waves compared to the wild-type Syt3, suggesting that both C2 domains of Syt3 in RGCs weigh similarly in regulating retinal waves. Second, to determine the role of Syt3 in RGCs in regulating the eye-specific segregation of the dLGN, we eliminated the expression of Syt3 by overexpressing the anti-sense Syt3 in RGCs using in vivo electroporation at P3. At P8, we examined the eye-specific segregation of the dLGN using axonal tracing and immunofluorescence staining. We found that the in vivo elimination of Syt3 in RGCs altered the eye-specific segregation of the dLGN and dampened stage II retinal waves in terms of wave duration and amplitude. These data suggest that Syt3 in developing RGCs is crucial for the eye-specific segregation of the dLGN. Consistently, the blockade of glutamate transmission in developing retinas regulates the eye-specific segregation of the dLGN, implying that glutamate release from RGCs within the retina is required for the correct eye-specific segregation of the dLGN. Together, our results support that Syt3 in developing RGCs can modulate patterned spontaneous activity in the retina and visual circuit refinement.