Neuronal Regulation of Vascular Development and Maturation in the Retina

项目摘要 出生后视网膜发育中神经胶质和血管成分之间的相互作用是 对血管的正常发育（血管生成）和成熟（血视网膜的建立）至关重要 屏障（BRB）]。虽然神经元或神经胶质细胞来源的信号，促进血管生成和BRB成熟 我们仍然不明白：a）神经元活动如何和B）哪些特定的 神经递质影响这些过程;和c）神经递质是否作用于内皮细胞（EC） 直接或间接通过星形胶质细胞或米勒细胞。浅丛脉管系统在 视网膜神经节细胞（RGC）自发胆碱能波和Opn 4+光活化（P1-P2） P9）。相比之下，深丛血管发育和BRB成熟跨越胆碱能和 神经元活动的突触能波（P10-P14），以及光感受器的突触能活动 作为Opn 4 + RGC的光活化。胆碱能波的短暂药物阻断延迟了深 神经丛血管生成和BRB成熟;然而，细胞外谷氨酸在这些过程中的作用是 未知在初步研究中，我们使用了两种小鼠品系来评估谷氨酸释放的影响。 a）在突触间隙中缺乏谷氨酸释放的VEGF 1-/-小鼠 和B）Gnat 1-/-小鼠，其中杆状光感受器缺乏光转导机制， 去极化，从而释放过量的谷氨酸。我们发现神经元活动依赖的谷氨酸 释放是深丛血管生成和BRB成熟的正调节剂。这些影响是通过 通过诱导Müller胶质细胞中的Norrin表达和EC中的Norrin/b-连环蛋白通路激活。基于这些 根据初步数据，我们假设细胞外谷氨酸水平被Müller细胞感知，而Müller细胞又反过来感知谷氨酸水平。 作为传感器，诱导血管生成因子和BRB形成因子的表达， 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，我们将研究如何 细胞外谷氨酸水平的调节[VEGF 1-/-小鼠（无谷氨酸释放）和Gnat 1-/-小鼠（高谷氨酸释放）] 谷氨酸释放）]调节视网膜血管生成和结构和功能的BRB的完整性， 视网膜。接下来，我们将测试Müller细胞是否感知细胞外谷氨酸水平，并通过诱导 血管生成因子和屏障生成因子的表达。我们将检查Müller细胞反应[细胞数量， GLAST（Slc 1a 3;一种主要的谷氨酸转运蛋白）、谷氨酰胺合成酶（一种负责 用于将谷氨酸转化为谷氨酰胺）和Norrin（一种由Müller细胞表达的血管生成因子）]。 /-和Gnat 1-/-小鼠。最后，我们将测试是否激活Norrin/b-连环蛋白信号，促进血管生成， 和BRB成熟，可以挽救VEGF 1-/-小鼠的缺陷。这些研究将提供一个机制 了解血管神经元突触活动如何调节血管发育和成熟， 视网膜和阐明谷氨酸兴奋性毒性如何影响神经退行性视网膜疾病的血管。

PROJECT SUMMARY Reciprocal interactions among neuroglial and vascular components in the developing postnatal retina are critical for proper vascular development (angiogenesis) and maturation [establishment of the blood-retina barrier (BRB)]. Although neuronal or glial cell-derived signals that promote angiogenesis and BRB maturation are being elucidated, we still do not understand: a) how neuronal activity and b) which specific neurotransmitter(s) influence these processes; and c) whether neurotransmitters act on endothelial cells (ECs) directly or indirectly, via astrocytes or Müller cells. The superficial plexus vasculature develops during the spontaneous cholinergic wave of neural activity and photoactivation of Opn4+ retinal ganglion cells (RGC) (P1- P9). In contrast, the deep plexus vascular development and BRB maturation span both cholinergic and glutamatergic waves of neuronal activity (P10-P14), glutamatergic synaptic activity of photoreceptors as well as photoactivation of Opn4+ RGCs. Transient pharmacological blockade of cholinergic waves delays deep plexus angiogenesis and BRB maturation; however, the role of extracellular glutamate in these processes is unknown. In preliminary studies, we have used two mouse strains to assess the effects of glutamate release on retinal angiogenesis and BRB maturation: a) Vglut1 -/- mice that lack glutamate release in the synaptic cleft and b) Gnat1-/- mice, in which rod photoreceptors lack the phototransduction machinery and remain depolarized, thus releasing excess glutamate. We have found that neuronal activity-dependent glutamate release is a positive regulator of deep plexus angiogenesis and BRB maturation. These effects are mediated by induction of Norrin expression in Müller glia and Norrin/b-catenin pathway activation in ECs. Based on these preliminary data, we hypothesize that extracellular 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 extracellular glutamate levels [Vglut1-/- mice (no glutamate release) and Gnat1-/- mice (high glutamate release)] regulates retinal angiogenesis and structural and functional BRB integrity in the developing retina. Next, we will test whether Müller cells sense extracellular glutamate levels and respond by inducing expression of angiogenic and barriergenic factors. We will examine Müller cell responses [cell number, expression of GLAST (Slc1a3; a major glutamate transporter), glutamine synthetase (an enzyme responsible for converting glutamate to glutamine) and Norrin (an angiogenesis factor expressed by Müller cells)] in Vglut1- /- and Gnat1-/- mice. Finally, we will test if activation of Norrin/b-catenin signalling, that promotes angiogenesis and BRB maturation, in ECs can rescue deficits of Vglut1-/- mice. These studies will provide a mechanistic understanding of how glutamatergic synaptic activity regulates vascular development and maturation in the retina and elucidate how glutamate excitotoxicity affects blood vessels in neurodegenerative retinal diseases.