The role of neuronal activity on retinal angiogenesis and blood-retina barrier (BRB) maturation

项目摘要 出生后视网膜的神经胶质细胞和血管成分之间的相互作用对于正常的视网膜发育至关重要。 血管生成和血-视网膜屏障（BRB）的建立。虽然神经元或神经胶质细胞来源 促进血管生成和BRB成熟的信号正在阐明，我们仍然不明白：a）如何 视网膜中的神经元突触活动影响这些过程; B）哪些神经递质有助于 这些过程;以及c）神经递质是否直接或间接作用于内皮细胞（EC）， 星形胶质细胞或米勒细胞。浅表神经丛脉管系统在自发性的 P0-P10时神经活动的胆碱能波和Opn 4+视网膜神经节细胞（RGC）的光活化。在 相反，深丛血管和BRB的发育跨越神经元的胆碱能波和多巴胺能波， 活动（P10-P14），光感受器的突触能活动（P10向前）以及光激活 Opn4+ RGC。胆碱能波的短暂药理学阻断损害深丛血管生成， BRB成熟;然而，细胞外谷氨酸在这些过程中的作用是未知的。初步 研究发现，我们主要使用三种小鼠品系来评估突触谷氨酸释放对 视网膜血管生成和BR B：a）Vnat 1-/-，缺乏谷氨酸释放，B）Gnat 1-/-，其中杆 光感受器保持去极化，从而释放过量的谷氨酸，和c）Chrnb 2-/-，其中胆碱能波是 P8时，脑电活动消失，脑电活动开始早。我们已经发现突触谷氨酸释放是一个积极的 深丛血管生成和BRB成熟的调节因子。这种效应是通过诱导 Müller胶质细胞中的Norrin和EC中的Norrin/b-连环蛋白通路激活。根据这些初步数据，我们 假设释放的谷氨酸水平被Müller细胞感知，Müller细胞反过来作为传感器工作， 诱导血管生成和BRB形成因子表达并促进深丛血管生成和BRB 成熟我们将通过三个目标来检验这一假设。首先，我们将研究谷氨酸的调节 水平[VEGF 1-/-小鼠（无谷氨酸释放）、Gnat 1-/-小鼠（高谷氨酸释放）和Chrnb 2-/-小鼠（早期 BRB波的起始）]调节视网膜血管生成以及结构和功能BRB完整性。接下来， 我们将确定Müller细胞是否通过诱导表达 血管生成因子和屏障生成因子。我们将检查Müller细胞反应[细胞数量、GLAST表达 （Slc 1a 3;一种主要的谷氨酸转运蛋白），谷氨酰胺合成酶（负责将谷氨酸转化为 谷氨酰胺）和Norrin（Müller细胞表达的主要Wnt配体）]。 最后，我们将测试是否激活内皮Norrin/β-连环蛋白信号，促进血管生成和BRB 成熟可以挽救VEGF 1-/-小鼠中的缺陷。这些研究将提供一个机械的理解， 神经元突触活性调节发育中视网膜的血管生成和BRB，并提供新的见解 兴奋性毒性对视网膜神经退行性疾病的影响。

PROJECT SUMMARY Reciprocal interactions among neuroglial and vascular components of the postnatal retina are critical for proper angiogenesis and the establishment of the blood-retinal barrier (BRB). Although neuronal or glial cells-derived signals that promote angiogenesis and BRB maturation are being elucidated, we still do not understand: a) how neuronal synaptic activity in the retina influences these processes; b) which neurotransmitter(s) contribute to these processes; and c) whether neurotransmitters act on endothelial cells (ECs) directly or indirectly, via astrocytes or Müller cells. The superficial plexus vasculature develops (P1-P8) during the spontaneous cholinergic wave of neural activity (P0-P10) and photoactivation of Opn4+ retinal ganglion cells (RGC). In contrast, the deep plexus vasculature and BRB development span cholinergic and glutamatergic waves of neural activity (P10-P14), glutamatergic synaptic activity of photoreceptors (P10 onwards) as well as photoactivation of Opn4+ RGCs. Transient pharmacological blockade of cholinergic wave impairs deep plexus angiogenesis and BRB maturation; however, the role of extracellular glutamate in these processes is unknown. In preliminary findings, we have primarily used three mouse strains to assess the effects of synaptic glutamate release on retinal angiogenesis and the BRB: a) Vglut1 -/-, that lack glutamate release, b) Gnat1-/-, in which rod photoreceptors remain depolarized, thus release excess glutamate, and c) Chrnb2-/-, where cholinergic wave is abolished and glutamatergic wave begins early (P8). We have found that synaptic glutamate release is a positive regulator of deep plexus angiogenesis and BRB maturation. This effect is mediated by inducing expression of Norrin in Müller glia and Norrin/b-catenin pathway activation in ECs. Based on these preliminary data, we hypothesize that released glutamate levels are sensed by Müller cells, which in turn operate as transducers to induce expression of angiogenic and BRB-forming factors and promote deep plexus angiogenesis and BRB maturation. We will test this hypothesis through three aims. First, we will examine how modulation of glutamate levels [Vglut1-/- mice (no glutamate release), Gnat1-/- mice (high glutamate release), and Chrnb2-/- mice (early onset of glutamatergic wave)] regulates retinal angiogenesis and structural and functional BRB integrity. Next, we will determine whether Müller cells respond to extracellular glutamate levels by inducing expression of angiogenic and barriergenic factors. We will examine Müller cell response [cell number, expression of GLAST (Slc1a3; a major glutamate transporter), glutamine synthetase (enzyme responsible for converting glutamate to glutamine) and Norrin (the major Wnt ligand expressed by Müller cells)] in Vglut1-/-, Gnat1-/- and Chrnb2-/- mice. Finally, we will test if activation of endothelial Norrin/b-catenin signalling that promotes angiogenesis and BRB maturation can rescue deficits in Vglut1-/- mice. These studies will provide a mechanistic understanding of how glutamatergic synaptic activity regulates angiogenesis and BRB in the developing retina and provide new insights into effects of excitotoxicity on neurodegenerative diseases in the retina.