Neuronal Regulation of Vascular Development and Maturation in the Retina

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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.