Neuronal Regulation of Vascular Development and Maturation in the Retina

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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.