Neuronal Regulation of Vascular Development and Maturation in the Retina

项目总结 神经元、神经胶质细胞和血管相关细胞之间的相互作用对正常的血管至关重要 早期发育(血管生成)和成熟[建立血-视网膜屏障(BRB)] 出生后视网膜。尽管促进血管生成和BRB的神经元或神经胶质细胞来源的信号 形成2是正在出现的，我们不知道神经元突触活动是如何的，一般情况下，以及哪些特定 神经递质(S)尤其参与了这些过程。我们也不知道神经递质是否起作用 直接作用于内皮细胞(ECs)或通过Müller胶质细胞间接作用。浅血管丛起源于 自发性胆碱能波的P_1~P_9。相比之下，深血管丛发育和BRB 成熟(P10-P14)发生在胆碱能波的末期，自发性和自发性 光感受器介导的谷氨酸能活性。一过性胆碱能波的药物阻断 延缓深部神经丛血管生成和BRB成熟；然而，细胞外谷氨酸在这些过程中的作用 进程未知。在初步研究中，我们使用了两个小鼠品系来评估 谷氨酸释放对视网膜血管生成和BRB成熟的影响：A)缺乏谷氨酸释放的小鼠 突触裂隙和b)Gnat1-/-小鼠在突触中结构性地释放谷氨酸。我们发现， 神经元活性依赖的谷氨酸释放是深丛血管生成和BRB的正向调节因子 成熟。这些作用是通过去甲肾上腺素诱导Müler神经胶质细胞和去甲肾上腺素/b-连环蛋白途径来实现的 内皮细胞中的激活。基于这些初步数据，我们假设细胞外谷氨酸水平是 被Müller细胞感知，进而作为转导分子诱导血管生成和BRB-1的表达 形成因素，促进深部神经丛血管生成和BRB成熟。我们将在以下方面测试这一假设 三个目标。首先，我们将研究细胞外谷氨酸水平的调节[Vher1-/-小鼠(无谷氨酸 和Gnat1-/-小鼠(高谷氨酸释放)]调节视网膜血管生成和结构和 发育中的视网膜中功能性BRB的完整性。我们还将研究谷氨酸和胆碱能 活动相互作用来调节这些过程。接下来，我们将测试Müler细胞是否感知细胞外 谷氨酸，并通过诱导血管生成因子和屏障因子的表达而做出反应。我们将检查穆勒 谷氨酸缺乏小鼠的细胞反应和去甲肾上腺素表达(血管生成因子) 神经元释放或Müler神经胶质细胞摄取。最后，我们将测试Norrin/b-catenin信号是否激活，即 促进血管生成和BRB成熟，在内皮细胞中可以修复Vher1-/-小鼠的缺陷。总体而言，我们的研究 将为谷氨酸能突触活动如何调节脑发育提供新的机制理解。 发育中视网膜深血管丛和BRB的成熟及谷氨酸的作用 兴奋性毒性可能影响神经退行性视网膜疾病的血管。

PROJECT SUMMARY Reciprocal interactions among neuronal, glial and vascular-associated cells are critical for proper vascular development (angiogenesis) and maturation [establishment of the blood-retina barrier (BRB)] in the early postnatal (P) retina. Although neuronal or glial cell-derived signals that promote angiogenesis and BRB formation2 are emerging, we do not understand how neuronal synaptic activity, in general, and which specific neurotransmitter(s), in particular, contribute to these processes. Nor do we know whether neurotransmitters act directly on endothelial cells (ECs) or indirectly via Müller glia. The superficial vascular plexus develops from P1-P9 during the spontaneous cholinergic wave. In contrast, the deep vascular plexus development and BRB maturation (P10-P14) occur at the end of the cholinergic wave and the onset of both spontaneous and photoreceptor-mediated glutamatergic activity. 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 that constitutively release glutamate in the synapse. 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 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 in 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. We will also examine how glutamatergic and cholinergic activity interact to regulate these processes. Next, we will test whether Müller cells sense extracellular glutamate and respond by inducing expression of angiogenic and barriergenic factors. We will examine Müller cell responses and Norrin expression (an angiogenesis factor) by Müller cells in mice deficient for glutamate release by neurons or uptake by Müller glia. 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. Overall, our studies will provide a novel mechanistic understanding of how glutamatergic synaptic activity regulates development of the deep vascular plexus and BRB maturation in the developing retina and elucidate how glutamate excitotoxicity may affect blood vessels in neurodegenerative retinal diseases.